1209 Citations
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp - HaDHR is a member of a small family of dehydrogenases containing a covalently attached FMN and the only member so far identified to date that does not exhibit substrate inhibition In this study we present the resolution crystal structure of HaDHR This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators Alanine was identified as the exit point of electrons from the Fe S cluster The enzyme was modified with a Ser Cys mutation to facilitate covalent attachment of a ferrocene moiety When modified ... More
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators. Less
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted compared to mesophilic orthologs In several cases the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation In the psychrophilic -amylase from an Antarctic bacterium the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature Here we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations ... More
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog. Less
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation T- domain responsible for the delivery of the catalytic domain into the cell Here we use a combination of computational constant-pH Molecular Dynamics simulations and experimental NMR circular dichroism and fluorescence spectroscopy along with the X-ray crystallography approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H Q H Q double mutant This replacement suppresses the acid-induced transition resulting in the retention of a more stable protein structure in solutions at ... More
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur. Less
We recently converted the GAF domain of NpR cyanobacteriochrome into near-infrared NIR fluorescent proteins FPs Unlike cyanobacterichrome which incorporates phycocyanobilin tetrapyrrole engineered NIR FPs bind biliverdin abundant in mammalian cells thus being the smallest scaffold for it Here we determined the crystal structure of the brightest blue-shifted protein of the series miRFP nano at resolution characterized its chromophore environment and explained the molecular basis of its spectral properties Using the determined structure we have rationally designed a red-shifted NIR FP termed miRFP nano with excitation at nm and emission at nm miRFP nano exhibits a small size of kDa enhanced ... More
We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities. Less
The gut microbiome is complex raising questions about the role of individual strains in the community Here we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid -dehydroxylation niche Omitting Clostridium scindens Cs and Clostridium hylemonae Ch eliminates secondary bile acid production and reshapes the community in a highly specific manner eight strains change in relative abundance by -fold In single-strain dropout communities Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent However Clostridium sporogenes increases -fold in the Cs but ... More
The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Less
Genome analysis of Pyrobaculum calidifontis revealed the presence of -glucosidase Pcal gene Structural analysis affirmed the presence of signature sequences of Type II -glucosidases in Pcal We have heterologously expressed the gene and produced recombinant Pcal in Escherichia coli Biochemical characteristics of the recombinant enzyme resembled to that of Type I -glucosidases instead of Type II Recombinant Pcal existed in a tetrameric form in solution and displayed highest activity at C and pH independent of any metal ions A short heat-treatment at C resulted in a increase in enzyme activity A slight structural shift was observed by CD spectrometry at ... More
Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry. Less
We have used a cohort of human induced pluripotent stem cell hiPSC lines to develop a laboratory-based drug screening platform to predict variable drug responses of potential clinical relevance Our approach is based on the findings that hiPSC lines reflect the genetic identity of the donor and that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state using high-throughput cell painting assays allowing detection of variable phenotypic responses to a wide range of clinically approved drugs across multiple ... More
We have used a cohort of human induced pluripotent stem cell (hiPSC) lines to develop a laboratory-based drug screening platform to predict variable drug responses of potential clinical relevance. Our approach is based on the findings that hiPSC lines reflect the genetic identity of the donor and that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins. We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state using high-throughput cell painting assays, allowing detection of variable phenotypic responses to a wide range of clinically approved drugs, across multiple disease areas. Furthermore, we provide information on mechanisms of drug-cell interactions underlying the observed variable responses by using quantitative proteomic analysis to compare sets of hiPSC lines that had been stratified objectively using cell painting data. We propose that information derived from comparative drug screening using curated libraries of hiPSC lines can help to increase the success rate of drug development pipelines and improve the delivery of safe new drugs suitable for a broader ethnic and gender diversity within human populations. Less
To address the limitation associated with degron based systems we have developed iTAG a synthetic tag based on IMiDs CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs CeLMoDs based tags Using structural and sequence analysis we systematically explored native and chimeric degron containing domains DCDs and evaluated their ability to induce degradation We identified the optimal chimeric iTAG DCD aa that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented hook effect of PROTAC-based systems We showed that iTAG can also induce target degradation by ... More
To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented “hook effect” of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome. Less
The high morbidity and mortality associated with SARS-CoV- infection the etiological agent of COVID- has had a major impact on global public health Significant progress has been made in the development of an array of vaccines and biologics however the emergence of SARS-CoV- variants and breakthrough infections are an ongoing major concern Furthermore there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV- and any emerging and re-emerging coronaviruses We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent ... More
The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 μM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1e and 1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies. Less
The Gs protein-coupled adenosine A A receptor A AAR represents an emerging drug target for cancer immunotherapy The clinical candidate Etrumadenant was developed as an A AAR antagonist with ancillary blockade of the A BAR subtype It constitutes a unique chemotype featuring a poly-substituted -amino- -phenyl- -triazolylpyrimidine core structure Herein we report two crystal structures of the A AAR in complex with Etrumadenant obtained with differently thermostabilized A AAR constructs This led to the discovery of an unprecedented interaction a hydrogen bond of T with the cyano group of Etrumadenant T is mutated in most A AAR constructs used for ... More
The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations. Less
Arc is an immediate early gene that regulates synaptic plasticity in glutamatergic neurons The formation of new long-term memories requires functioning Arc protein Arc is both a protein interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like capsids that transfect nearby cells Relatively little is known about the structure of the mammalian Arc protein It consists of mainly -helical structures that make up the N- and Cterminal domain which are connected by a flexible linker and flanked by flexible N- and Cterminal tails Arc is found in many oligomeric states ranging from dimers ... More
Arc is an immediate early gene that regulates synaptic plasticity in glutamatergic neurons. The
formation of new long-term memories requires functioning Arc protein. Arc is both a protein
interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like
capsids that transfect nearby cells. Relatively little is known about the structure of the
mammalian Arc protein. It consists of mainly α-helical structures that make up the N- and Cterminal
domain, which are connected by a flexible linker and flanked by flexible N- and Cterminal
tails. Arc is found in many oligomeric states ranging from dimers to the predicted 140-
subunit capsids. This study aimed to solve the full-length structure of dimeric mammalian Arc
using X-ray crystallography and single-particle cryo-EM. Nanobodies that bind to Arc with
high affinity were used to stabilize and enlarge the dimeric complex. Structural information
about the Arc-nanobody complex was gathered using SAXS and compared with structures
predicted using AlphaFold. The results show that AlphaFold struggles to predict structures that
match the low-resolution structures of Arc in solution obtained from SAXS, likely due to Arc’s
structural flexibility and tendency to oligomerize. The study has also provided insight into the
binding dynamics of these nanobodies to Arc and highlighted their many uses in addition to
structural chaperones. Optimization of the sample preparation and data collection were
performed for the use in single-particle cryo-EM to solve the dimeric structure of full-length
Arc, although the data collection and processing have not been finished as of this moment. This
data could provide valuable new structural information about Arc, which will help better the
understanding of Arc’s functions and roles in disease. Less
formation of new long-term memories requires functioning Arc protein. Arc is both a protein
interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like
capsids that transfect nearby cells. Relatively little is known about the structure of the
mammalian Arc protein. It consists of mainly α-helical structures that make up the N- and Cterminal
domain, which are connected by a flexible linker and flanked by flexible N- and Cterminal
tails. Arc is found in many oligomeric states ranging from dimers to the predicted 140-
subunit capsids. This study aimed to solve the full-length structure of dimeric mammalian Arc
using X-ray crystallography and single-particle cryo-EM. Nanobodies that bind to Arc with
high affinity were used to stabilize and enlarge the dimeric complex. Structural information
about the Arc-nanobody complex was gathered using SAXS and compared with structures
predicted using AlphaFold. The results show that AlphaFold struggles to predict structures that
match the low-resolution structures of Arc in solution obtained from SAXS, likely due to Arc’s
structural flexibility and tendency to oligomerize. The study has also provided insight into the
binding dynamics of these nanobodies to Arc and highlighted their many uses in addition to
structural chaperones. Optimization of the sample preparation and data collection were
performed for the use in single-particle cryo-EM to solve the dimeric structure of full-length
Arc, although the data collection and processing have not been finished as of this moment. This
data could provide valuable new structural information about Arc, which will help better the
understanding of Arc’s functions and roles in disease. Less
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy Here we introduce scEC T-seq s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells In this protocol a single cell ... More
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival. A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity. Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy. Here, we introduce scEC&T-seq ( s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells. In this protocol, a single cell’s DNA is separated from its polyadenylated RNA as described by Macaulay et al. (2015) 1 . This is followed by removal of linear DNA through exonuclease digestion and further enrichment of circular DNA by rolling circle amplification with φ29 polymerase 2-4 . The separated mRNA from the same cell is processed using on-bead Smart-seq2 1 . The duration of the entire procedure from cell sorting to library preparation is approximately 8 days. Our scEC&T-seq protocol has been validated in single cancer cells from neuroblastoma cell lines and primary tumors, and in normal single T-cells isolated from patient’s blood. Besides identifying large, oncogene-containing circular DNAs in cancer cells, our method also captures other smaller circular DNAs, which have been previously described in both cancer and non-malignant cells 5 . We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development. Less
Cytotoxic-T-lymphocyte CTL mediated control of HIV- is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I HLA-I However the extent to which the presenting HLA allele contributes to this process is unknown Here we examine the CTL response to QW a highly networked epitope presented by the disease-protective HLA-B and disease-neutral HLA-B Despite robust targeting of QW in persons expressing either allele T cell receptor TCR cross-recognition of the naturally occurring variant QW S T is consistently reduced when presented by HLA-B but not by HLA-B Crystal structures show substantial conformational changes from QW -HLA to QW S T-HLA ... More
Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design. Less
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development however the molecular mechanisms underlying this phenomenon in mammals remain poorly described Here we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days and and compared the species using a framework for time-resolved single-cell differentiation-flows analysis We find convergence toward similar cell-state compositions at E supported by the quantitatively conserved expression of transcription factors despite divergence in surrounding trophoblast and hypoblast signaling However we ... More
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals. Less
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool Room-temperature data collection also enables time-resolved experiments In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines room-temperature methodology is less mature Here the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described and a highly efficient pipeline from protein sample to ... More
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention. Less
Nowadays the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts Only a few commercial microfluidic systems for enzyme analysis are currently available and the transformation of numerous published prototypes ... More
Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development. Less
Latrophilins ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects lethality and sterility across multiple model organisms However their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown Here we identified a cell-surface receptor TOL- the sole Toll-like receptor in C elegans as a novel ligand for the C elegans Latrophilin LAT- The extracellular lectin domain of LAT- directly binds to the second leucine-rich repeat domain of TOL- The highresolution crystal structure and the cryo-EM density map of the LAT- TOL- ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by ... More
Latrophilins/ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects, lethality, and sterility across multiple model organisms. However, their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown. Here, we identified a cell-surface receptor, TOL-1, the sole Toll-like receptor in C. elegans, as a novel ligand for the C. elegans Latrophilin, LAT-1. The extracellular lectin domain of LAT-1 directly binds to the second leucine-rich repeat domain of TOL-1. The highresolution crystal structure and the cryo-EM density map of the LAT-1–TOL-1 ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by a large interface. CRISPR/Cas9-mediated mutation of key interface residues selectively disrupted the endogenous LAT-1–TOL-1 interaction in C. elegans, leading to partial sterility, lethality, and malformed embryos. Thus, TOL-1 binding to LAT-1 represents a receptor-ligand axis essential for animal morphogenesis. Less
Despite advances in high-dimensional cellular analysis the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways
In the DarTG toxin-antitoxin system the DarT toxin ADP-ribosylates single-stranded DNA ssDNA which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG DarG also binds DarT but the role of these interactions in DarT neutralization is unknown Here we report that the C-terminal domain of DarG DarG toxin-binding domain DarGTBD interacts with DarT to form a stoichiometric heterodimeric complex We determined the resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex The comparative structural analysis reveals that DarGTBD interacts with ... More
In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions. Less
Training artificial intelligence AI systems to perform autonomous experiments would vastly increase the throughput of microbiology however few microbes have large enough datasets for training such a system In the present study we introduce BacterAI an automated science platform that maps microbial metabolism but requires no prior knowledge BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots The agent then distils its findings into logical rules that can be interpreted by human scientists We use BacterAI to learn the amino acid requirements for two oral streptococci Streptococcus gordonii and Streptococcus sanguinis We then show ... More
Training artificial intelligence (AI) systems to perform autonomous experiments would vastly increase the throughput of microbiology; however, few microbes have large enough datasets for training such a system. In the present study, we introduce BacterAI, an automated science platform that maps microbial metabolism but requires no prior knowledge. BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots. The agent then distils its findings into logical rules that can be interpreted by human scientists. We use BacterAI to learn the amino acid requirements for two oral streptococci: Streptococcus gordonii and Streptococcus sanguinis. We then show how transfer learning can accelerate BacterAI when investigating new environments or larger media with up to 39 ingredients. Scientific gameplay and BacterAI enable the unbiased, autonomous study of organisms for which no training data exist. Less
Proteorhodopsins PRs bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins they play a significant role in life on the Earth A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name mirror proteorhodopsins from Sphingomonas paucimobilis SpaR shows cavity gate architecture of ... More
Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name “mirror proteorhodopsins”, from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest. Less
SARS-CoV- and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including CL protease PL protease and RNA dependent RNA polymerase This dissertation summarizes the research findings related to CL protease inhibitors of SARS-CoV- and MERS-CoV A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor- CL protease complexes The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency Multiple inhibitors ... More
SARS-CoV-2 and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population. Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including 3CL protease, PL protease and RNA dependent RNA polymerase.
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
Influenza virus IV causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars Several influenza pandemics have occurred during the last century and estimated to have caused million deaths There are four genera of IV A IVA B IVB C IVC and D IVD with IVA being the most virulent to the human population Hemagglutinin HA is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell Here we have characterised the high-resolution structures of seven IVA HAs with one ... More
Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines. Less
The microbial guild coupling anammox and nitrite nitrate-dependent anaerobic methane oxidation n-DAMO is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater Here metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater respectively We find that different nitrogen loadings i e vs kg N m d lead to different combinations of anammox bacteria and anaerobic methanotrophs Candidatus Methanoperedens and Candidatus Methylomirabilis which play primary roles for ... More
The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs (“Candidatus Methanoperedens” and “Candidatus Methylomirabilis”), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to “Ca. Methylomirabilis” are recovered, while one surprisingly expresses nitrate reductases, which we designate as “Ca. Methylomirabilis nitratireducens” representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of “Ca. Methylomirabilis” bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea. Less
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes It has two different enzymatic activities microbial cell walls destruction muramidase activity and dissolution of the stabilized fibrin isopeptidase activity Both activities are known to be inhibited by sodium chloride at near physiological concentrations but the structural basis remains unknown Here we present two crystal structures of destabilase including a -resolution structure in complex with sodium ion Our structures reveal the location of sodium ion between Glu Asp residues which were previously recognized as a glycosidase active site While sodium coordination with these amino acids may ... More
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development. Less
Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge which also presents opportunities for chemical and synthetic biology Here we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli E coli To do this we combine de novo -helical sequences intrinsically disordered linkers and client proteins in ... More
Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems. The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge, which also presents opportunities for chemical and synthetic biology. Here, we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli (E. coli). To do this, we combine de novo α-helical sequences, intrinsically disordered linkers, and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from diffusion-limited assemblies to dynamic condensates. The designs are characterised in cells and in vitro using biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair in E. coli. Less
Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell cell communication multicellular cohabitation principles and the higher resilience of microorganisms in a biofilm against antibiotics Biofilms of the nonpathogenic gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection Its major extracellular matrix protein components are TasA and TapA The nature of TasA filaments has been of debate and several forms amyloidic and non-Thioflavin T-stainable have been observed Here we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments By analytical ... More
Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell–cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.
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Described herein are methods for stratifying and evaluating melanoma treatment response in a subject using single-cell RNA sequencing scRNA-seq and a two-step deconvolution analysis and optionally administering a treatment depending on the results Embodiment described herein are methods for stratifying and evaluating melanoma treatment response in a subject based on single cell or bulk RNA sequencing bulk transcriptome profiling and or transcript counting and a two-step deconvolution analysis and optionally administering a treatment depending on the results
Transporters of the Nramp Natural resistance-associated macrophage protein family import divalent transition metal ions into cells of most organisms By supporting metal homeostasis Nramps prevent diseases and disorders related to metal insufficiency or overload Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site We present high-resolution structures of Deinococcus radiodurans Dra Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate Mn across conformations and by conserved networks of polar residues lining the inner and outer gates In addition a ... More
Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps. Less
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine which is tightly bound in the protein inner cavity The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed many features of their functioning are yet unclear In particular which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role ... More
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the “catalytic function” by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins. Less
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source thereby reducing the risk of kidney stone formation in host animals The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates Here we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations occluded and outward-facing states The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate The occluded pocket can accommodate ... More
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis. Less
Pseudomonas aeruginosa is a multi-drug resistant human opportunistic pathogen If left untreated P aeruginosa can cause severe to life-threatening infections in people with burns cystic fibrosis and in immunocompromised patients During chronic infections P aeruginosa primarily co-ordinates virulence in the host through a cell-to-cell communication mechanism called quorum sensing QS There are three key QS systems in P aeruginosa responsible for driving global changes in virulence gene expression the las rhl and pqs systems Each of the las rhl and pqs systems rely on a receptor-autoinducer relationship these receptor-autoinducer complexes are LasR-OdDHL RhlR-BHL and PqsR-PQS respectively When the receptors LasR ... More
Pseudomonas aeruginosa is a multi-drug resistant, human opportunistic pathogen. If left untreated, P. aeruginosa can cause severe to life-threatening infections in people with burns, cystic fibrosis, and in immunocompromised patients. During chronic infections, P. aeruginosa primarily co-ordinates virulence in the host through a cell-to-cell communication mechanism called quorum sensing (QS). There are three key QS systems in P. aeruginosa responsible for driving global changes in virulence gene expression: the las, rhl, and pqs systems. Each of the las, rhl, and pqs systems rely on a receptor-autoinducer relationship: these receptor-autoinducer complexes are LasR-OdDHL, RhlR-BHL, and PqsR-PQS, respectively. When the receptors (LasR, RhlR, PqsR) bind with their cognate autoinducer (OdDHL, BHL, PQS, respectively), they act as transcription factors that ultimately stimulate the expression of hundreds of virulence-associated genes. The influence these QS systems have on the expression of virulence determinants has led to decades of scientific research focusing on the characterisation of these regulators. Although LasR and PqsR have been structurally elucidated, the RhlR crystal structure has long eluded characterisation and has been highly sought after due to its obvious potential as a therapeutic target.
In a collaborative research effort, I helped to identify ten additional proteins as putative binding partners of the pqs autoinducer, PQS. Four of the ten proteins identified were the cyanide synthase (HcnC), a putative protease (PfpI), a phenazine biosynthetic protein (PhzD1), and the QS regulator RhlR. For this PhD project, I aimed to structurally and biochemically characterise these four proteins to, in part, confirm their proposed interaction with PQS. A novel ligand (benzoic acid) was discovered bound in the active site of PhzD1 (crystal structure solved to 1.1 Å). Additionally, the crystal structure for PfpI was resolved at 1.4 Å resolution. The PfpI tertiary and quaternary structures obtained in this study suggested a possible role in electrophile detoxification, a hypothesis which I confirmed in vitro using 1D NMR. To complement the novel PfpI structural and biochemical data, I generated and confirmed “clean” pfpI deletion mutants for phenotypic and ‘omic analyses. I observed discrepancies in phenotypes between the pfpI deletion mutant and the pfpI transposon mutants previously reported in the published literature, which I sought to reconcile through subsequent whole genome sequencing (WGS) of these previously published strains. WGS of the pfpI transposon mutants revealed a plethora of unexpected mutations elsewhere in the genome, which likely contribute to many of the reported phenotypes. The “clean” deletion mutant that I generated harboured no significant additional mutations. Proteomic profiling of the pfpI deletion mutant exhibited altered protein expression in systems involved in Type VI secretion, motility, and metabolism.
Overall, the work presented in this dissertation further illustrates the intractability of purifying the QS transcriptional regulator, RhlR. I report benzoic acid to be a novel binding partner for the phenazine biosynthetic protein, PhzD1. Phenotypic analyses of pfpI mutants and consequent WGS highlight the need for rigorous strain validation when using transposon mutant libraries. Using the PfpI structural data I obtained during this study, I hypothesised and confirmed a novel detoxification role for PfpI in P. aeruginosa. Lastly, proteomic analysis of a pfpI deficient mutant revealed global dysregulation of key biological processes. Less
In a collaborative research effort, I helped to identify ten additional proteins as putative binding partners of the pqs autoinducer, PQS. Four of the ten proteins identified were the cyanide synthase (HcnC), a putative protease (PfpI), a phenazine biosynthetic protein (PhzD1), and the QS regulator RhlR. For this PhD project, I aimed to structurally and biochemically characterise these four proteins to, in part, confirm their proposed interaction with PQS. A novel ligand (benzoic acid) was discovered bound in the active site of PhzD1 (crystal structure solved to 1.1 Å). Additionally, the crystal structure for PfpI was resolved at 1.4 Å resolution. The PfpI tertiary and quaternary structures obtained in this study suggested a possible role in electrophile detoxification, a hypothesis which I confirmed in vitro using 1D NMR. To complement the novel PfpI structural and biochemical data, I generated and confirmed “clean” pfpI deletion mutants for phenotypic and ‘omic analyses. I observed discrepancies in phenotypes between the pfpI deletion mutant and the pfpI transposon mutants previously reported in the published literature, which I sought to reconcile through subsequent whole genome sequencing (WGS) of these previously published strains. WGS of the pfpI transposon mutants revealed a plethora of unexpected mutations elsewhere in the genome, which likely contribute to many of the reported phenotypes. The “clean” deletion mutant that I generated harboured no significant additional mutations. Proteomic profiling of the pfpI deletion mutant exhibited altered protein expression in systems involved in Type VI secretion, motility, and metabolism.
Overall, the work presented in this dissertation further illustrates the intractability of purifying the QS transcriptional regulator, RhlR. I report benzoic acid to be a novel binding partner for the phenazine biosynthetic protein, PhzD1. Phenotypic analyses of pfpI mutants and consequent WGS highlight the need for rigorous strain validation when using transposon mutant libraries. Using the PfpI structural data I obtained during this study, I hypothesised and confirmed a novel detoxification role for PfpI in P. aeruginosa. Lastly, proteomic analysis of a pfpI deficient mutant revealed global dysregulation of key biological processes. Less
Background Lower limb peripheral artery disease PAD is the main risk of diabetes mellitus which result to high mortality rate Approximately of patients who receive several treatments have passed away or lost limbs at a year s follow-up Secretome of hypoxia mesenchymal stem cells S-MSCs contains several active soluble molecules from hypoxia MSCs H-MSCs that capable inducing anti-inflammatory and vascular regeneration in PAD Objective In this study we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats Methods The PAD was established by the incision from the groin to the inner thigh and ... More
Background:
Lower limb peripheral artery disease (PAD) is the main risk of diabetes mellitus which result to high mortality rate. Approximately, 50% of patients who receive several treatments have passed away or lost limbs at a year’s follow-up. Secretome of hypoxia mesenchymal stem cells (S-MSCs) contains several active soluble molecules from hypoxia MSCs (H-MSCs) that capable inducing anti-inflammatory and vascular regeneration in PAD.
Objective:
In this study, we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats.
Methods:
The PAD was established by the incision from the groin to the inner thigh and distal ligation of femoral arteries in rats with diabetes. Rats were administered with 200 µL and 400 µL S-MSCs that successfully filtrated using tangential flow filtration (TFF) system based on various molecular weight cut-off categories intravenously. ELISA assay was used to analyze the cytokines and growth factors contained in S-MSCs. Tarlov score were examined at day 1, 3, 5, 7, 10 and 14. The rats were sacrificed at day 14 and muscle tissues were collected for immunohistochemistry (IHC) and gene expression analysis.
Results:
ELISA assay showed that S-MSCs provides abundant level of VEGF, PDGF, bFGF, IL-10 and TGFβ. In vivo administration of S-MSCs remarkably enhanced the Tarlov score. S-MSCs improved angiogenesis through enhancing VEGF gene expression and significantly increasing CD31 positive area in muscle tissue of PAD diabetic rats.
Conclusion:
Our findings suggest that S-MSCs could improves dynamic function and angiogenesis in PAD diabetic rats. Less
Lower limb peripheral artery disease (PAD) is the main risk of diabetes mellitus which result to high mortality rate. Approximately, 50% of patients who receive several treatments have passed away or lost limbs at a year’s follow-up. Secretome of hypoxia mesenchymal stem cells (S-MSCs) contains several active soluble molecules from hypoxia MSCs (H-MSCs) that capable inducing anti-inflammatory and vascular regeneration in PAD.
Objective:
In this study, we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats.
Methods:
The PAD was established by the incision from the groin to the inner thigh and distal ligation of femoral arteries in rats with diabetes. Rats were administered with 200 µL and 400 µL S-MSCs that successfully filtrated using tangential flow filtration (TFF) system based on various molecular weight cut-off categories intravenously. ELISA assay was used to analyze the cytokines and growth factors contained in S-MSCs. Tarlov score were examined at day 1, 3, 5, 7, 10 and 14. The rats were sacrificed at day 14 and muscle tissues were collected for immunohistochemistry (IHC) and gene expression analysis.
Results:
ELISA assay showed that S-MSCs provides abundant level of VEGF, PDGF, bFGF, IL-10 and TGFβ. In vivo administration of S-MSCs remarkably enhanced the Tarlov score. S-MSCs improved angiogenesis through enhancing VEGF gene expression and significantly increasing CD31 positive area in muscle tissue of PAD diabetic rats.
Conclusion:
Our findings suggest that S-MSCs could improves dynamic function and angiogenesis in PAD diabetic rats. Less
Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes deaths annually in the United States prompting a need for vaccine development In addition to the important toxins TcdA and TcdB binary toxin CDT plays a significant role in the pathogenesis of certain C difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells However the mechanisms of CDT neutralization by antibodies have not been studied limiting our understanding of key epitopes for CDT antigen design Therefore we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically Here - and - resolution X-ray ... More
Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb—the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol—showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics. Less
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors GPCRs A photon is absorbed by the -cis retinal chromophore of rhodopsin which isomerizes within femtoseconds to the all-trans conformation thereby initiating the cellular signal transduction processes that ultimately lead to vision However the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding ... More
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation. Less
Background With an increasing interest in the manipulation of methane produced from livestock cultivation the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with low-methane emitters Previously marsupial species were shown to be enriched for novel lineages of Methanocorpusculum as well as Methanobrevibacter Methanosphaera and Methanomassiliicoccales Despite sporadic reports of Methanocorpusculum from stool samples of various animal species there remains little information on the impacts of these methanogens on their hosts Results Here we characterise novel host-associated species of Methanocorpusculum to explore unique host-specific genetic factors and their associated metabolic potential We performed comparative analyses on Methanocorpusculum ... More
Background
With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.
Results
Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).
Conclusions
Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated. Less
With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.
Results
Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).
Conclusions
Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated. Less
Background In aquatic ecosystems the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment Nonetheless finding microbes with profound impacts on fish s performance out of thousands of candidate species remains a major challenge Methods We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background environmental water By targeting eel-aquaculture-tank microbiomes as model systems we reconstructed the population dynamics of the bacterial and archaeal species strains across days Results Due to the remarkable increase decrease of constituent microbial population densities ... More
Background
In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish’s performance out of thousands of candidate species remains a major challenge.
Methods
We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days.
Results
Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels’ activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels’ activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed “core microbiomes” with potentially positive impacts on eels.
Conclusions
Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes. Less
In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish’s performance out of thousands of candidate species remains a major challenge.
Methods
We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days.
Results
Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels’ activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels’ activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed “core microbiomes” with potentially positive impacts on eels.
Conclusions
Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes. Less
The modified nucleosides -deoxy- -cyano- and -deoxy- -amido- -deazaguanosine dPreQ and dADG respectively recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster the latter of which encodes proteins that comprise the elaborate Dpd restriction modification system present in diverse bacteria Recent genetic studies implicated the dpdA dpdB and dpdC genes as encoding proteins necessary for DNA modification with dpdD dpdK contributing to the restriction phenotype Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo the elucidation of the roles of each protein ... More
The modified nucleosides 2′-deoxy-7-cyano- and 2′-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction–modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD–dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ0), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ0 into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ0-modified DNA to ADG-modified DNA. Less
A rare coding variant in PLC P R expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease LOAD and therefore activation of wild-type PLC has been suggested as a potential therapeutic target for the prevention and treatment of LOAD Additionally PLC has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLC activity have been identified Here pharmacological inhibition may provide a therapeutic effect In order to facilitate our ... More
A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various “turn-on” fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening. Less
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells NSPCs that give rise to the neurons oligodendrocytes and astrocytes of the mature brain Functional study of these cell types has been hampered by a lack of precise purification methods We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers CD THY lo cells were enriched for radial glia which robustly engrafted and differentiated into all three neural lineages in the mouse brain THY hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial ... More
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24−THY1−/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1−/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA− bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment. Less
Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families broadly referred to as E E and E results in the covalent linking of ubiquitin to a target protein While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein Ser Thr and Tyr can also be susceptible to ubiquitination through an oxyester bond Once ubiquitinated multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins Ubiquitination regulates multiple cellular ... More
Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families, broadly referred to as E1, E2 and E3, results in the covalent linking of ubiquitin to a target protein. While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein, Ser, Thr, and Tyr can also be susceptible to ubiquitination through an oxyester bond. Once ubiquitinated, multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins. Ubiquitination regulates multiple cellular processes, but it is best known as a modification that targets proteins for proteasomal degradation following the formation poly-ubiquitin chains linked through lysine 48 or 63 of ubiquitin. Dysregulation of ubiquitination has been associated with multiple types of cancer and efforts have been carried out to develop technologies that lead to the identification of inhibitors of the enzymes involved in the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor auto-ubiquitination of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a robust signal window with a robust average Z’ factor of 0.76. From a validatory screening experiment we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. Additionally, we have expanded the system to study deubiquitinases such as USP28 that lead to reduction of FRET due to hydrolysis of fluorescent poly-Ub chains. Less
Analyzing proteins from single cells by tandem mass spectrometry MS has recently become technically feasible While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design sample preparation data acquisition and data analysis We expect that broadly accepted community guidelines and standardized metrics will enhance rigor data quality and alignment between laboratories Here we propose best practices quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics
Tyrosine kinases TKs play essential roles in signaling processes that regulate cell survival migration and proliferation Dysregulation of tyrosine kinases underlies many disorders including cancer cardiovascular and developmental diseases as well as pathologies of the immune system Ack and Brk are nonreceptor tyrosine kinases NRTKs best known for their roles in cancer Here we have biochemically characterized novel Ack and Brk mutations identified in patients with systemic lupus erythematosus SLE These mutations are the first SLE-linked polymorphisms found among NRTKs We show that two of the mutants are catalytically inactive while the other three have reduced activity To understand the ... More
Tyrosine kinases (TKs) play essential roles in signaling processes that regulate cell survival, migration, and proliferation. Dysregulation of tyrosine kinases underlies many disorders, including cancer, cardiovascular and developmental diseases, as well as pathologies of the immune system. Ack1 and Brk are nonreceptor tyrosine kinases (NRTKs) best known for their roles in cancer. Here, we have biochemically characterized novel Ack1 and Brk mutations identified in patients with systemic lupus erythematosus (SLE). These mutations are the first SLE-linked polymorphisms found among NRTKs. We show that two of the mutants are catalytically inactive, while the other three have reduced activity. To understand the structural changes associated with the loss-of-function phenotype, we solved the crystal structure of one of the Ack1 kinase mutants, K161Q. Furthermore, two of the mutated residues (Ack1 A156 and K161) critical for catalytic activity are highly conserved among other TKs, and their substitution in other members of the kinase family could have implications in cancer. In contrast to canonical gain-of-function mutations in TKs observed in many cancers, we report loss-of-function mutations in Ack1 and Brk, highlighting the complexity of TK involvement in human diseases. Less
We report the structural biochemical and functional characterization of the product of gene PA from Pseudomonas aeruginosa PAO The protein termed Pa Dps adopts the Dps subunit fold and oligomerizes into a nearly spherical -mer quaternary structure at pH or in the presence of divalent cations at neutral pH and above The -Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer coordinated by conserved His Glu and Asp residues In vitro the di-iron centers catalyze the oxidation of Fe utilizing H O not O as an oxidant suggesting Pa Dps functions to aid P aeruginosa ... More
We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps. Less
Since the discovery of penicillin over a century ago secondary metabolites from all kingdoms of life have proven to be of high medical value One class of proteins prevalent in the production of secondary metabolites are polyketide synthases PKSs Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche To contribute to such an advantage a significant proportion of polyketides are active against pro- and eukaryotic microorganisms Type I PKSs are giant multienzyme proteins employing ... More
Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product.
In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action.
The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner.
In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates.
The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable. Less
In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action.
The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner.
In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates.
The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable. Less
Pure bacterial cultures remain essential for detailed experimental and mechanistic studies in microbiome research and traditional methods to isolate individual bacteria from complex microbial ecosystems are labor-intensive difficult-to-scale and lack phenotype genotype integration Here we describe an open-source high-throughput robotic strain isolation platform for the rapid generation of isolates on demand We develop a machine learning approach that leverages colony morphology and genomic data to maximize the diversity of microbes isolated and enable targeted picking of specific genera Application of this platform on fecal samples from humans yields personalized gut microbiome biobanks totaling isolates that represented of all abundant taxa ... More
Pure bacterial cultures remain essential for detailed experimental and mechanistic studies in microbiome research, and traditional methods to isolate individual bacteria from complex microbial ecosystems are labor-intensive, difficult-to-scale and lack phenotype–genotype integration. Here we describe an open-source high-throughput robotic strain isolation platform for the rapid generation of isolates on demand. We develop a machine learning approach that leverages colony morphology and genomic data to maximize the diversity of microbes isolated and enable targeted picking of specific genera. Application of this platform on fecal samples from 20 humans yields personalized gut microbiome biobanks totaling 26,997 isolates that represented >80% of all abundant taxa. Spatial analysis on >100,000 visually captured colonies reveals cogrowth patterns between Ruminococcaceae, Bacteroidaceae, Coriobacteriaceae and Bifidobacteriaceae families that suggest important microbial interactions. Comparative analysis of 1,197 high-quality genomes from these biobanks shows interesting intra- and interpersonal strain evolution, selection and horizontal gene transfer. This culturomics framework should empower new research efforts to systematize the collection and quantitative analysis of imaging-based phenotypes with high-resolution genomics data for many emerging microbiome studies. Less
Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication DNA repair and telomere maintenance Due to their essential role they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited Here we present a simple and versatile real-time exonuclease assay based on -aminopurine an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both and DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor ... More
Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair, and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited. Here, we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3′ and 5′ DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the Mycobacterium tuberculosis PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high-throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance. Less
G-protein metallochaperone MeaB in bacteria methylmalonic aciduria type A MMAA in humans is responsible for facilitating the delivery of adenosylcobalamin AdoCbl to methylmalonyl-CoA mutase MCM the only AdoCbl-dependent enzyme in humans Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids Here we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate GTP analog guanosine- - -methyleno triphosphate GMPPCP with the Cbl-binding domain of its target mutase enzyme MeMCMcbl This structure provides an explanation for the stimulation of the ... More
G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5′-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery. Less
Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available providing a critical understanding of macromolecular architecture These methods require crystallization of the target molecule which remains a primary bottleneck in crystal-based structure determination The National High-Throughput Crystallization Center at Hauptman Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions This paper will describe the lessons learned from over years of operation of our high-throughput crystallization services The current experimental pipelines instrumentation imaging capabilities and software for ... More
Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman–Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on. Less
Subcutaneous SC administration is a desired route for monoclonal antibodies mAbs However formulating mAbs for small injection volumes at high concentrations with suitable stability and injectability is a significant challenge Here this work presents a platform technology that combines the stability of crystalline antibodies with injectability and tunability of soft hydrogel particles Composite alginate hydrogel particles are generated via a gentle centrifugal encapsulation process which avoids use of chemical reactions or an external organic phase Crystalline suspension of anti-programmed cell death protein PD- antibody pembrolizumab is utilized as a model therapeutic antibody Crystalline forms of the mAb encapsuled in the ... More
Subcutaneous (SC) administration is a desired route for monoclonal antibodies (mAbs). However, formulating mAbs for small injection volumes at high concentrations with suitable stability and injectability is a significant challenge. Here, this work presents a platform technology that combines the stability of crystalline antibodies with injectability and tunability of soft hydrogel particles. Composite alginate hydrogel particles are generated via a gentle centrifugal encapsulation process which avoids use of chemical reactions or an external organic phase. Crystalline suspension of anti-programmed cell death protein 1 (PD-1) antibody (pembrolizumab) is utilized as a model therapeutic antibody. Crystalline forms of the mAb encapsuled in the hydrogel particles lead to stable, high concentration, and injectable formulations. Formulation concentrations as high as 315 mg mL−1 antibody are achieved with encapsulation efficiencies in the range of 89–97%, with no perceivable increase in the number of antibody aggregates. Bioanalytical studies confirm superior maintained quality of the antibody in comparison with formulation approaches involving organic phases and chemical reactions. This work illustrates tuning the alginate particles’ disintegration by using partially oxide alginates. Crystalline mAb-laden particles are evaluated for their biocompatibility using cell-based in vitro assays. Furthermore, the pharmacokinetics (PK) of the subcutaneously delivered human anti-PD-1 mAb in crystalline antibody-laden alginate hydrogel particles in Wistar rats is evaluated. Less