1209 Citations
To enhance the bioavailability of poorly soluble therapeutics enabling formulations that generate supersaturation are currently of great interest There is limited knowledge of how the gastrointestinal environment can influence the complex phase behavior of these systems in particular crystallization Simulated media are generally used to mimic physiologically relevant fluids although their predictability remains unknown for crystallizing systems since they are simplified models of the gastrointestinal fluids The purpose of this study was to evaluate and compare how different simulated media as well as aspirated intestinal fluid impact the phase behavior of supersaturated solutions of two poorly soluble compounds atazanavir and ... More
To enhance the bioavailability of poorly soluble therapeutics, enabling formulations that generate supersaturation are currently of great interest. There is limited knowledge of how the gastrointestinal environment can influence the complex phase behavior of these systems, in particular, crystallization. Simulated media are generally used to mimic physiologically relevant fluids, although their predictability remains unknown for crystallizing systems since they are simplified models of the gastrointestinal fluids. The purpose of this study was to evaluate and compare how different simulated media, as well as aspirated intestinal fluid, impact the phase behavior of supersaturated solutions of two poorly soluble compounds, atazanavir and posaconazole, in fasted-state conditions. The onset of nucleation and progression of crystallization were found to be highly medium dependent. In the aspirated fluid, the crystallization kinetics for both compounds was significantly reduced compared to commercial simulated media. The use of simple buffers or current simulated fluids as surrogates for intestinal fluids appears to require further verification when attempting to predict crystallization kinetics of supersaturated solutions, based on the observed lack of correlation between commercial media and human fluids. The findings highlight the importance of carefully considering the composition of in vitro testing media for assessing crystallization kinetics, particularly in the context of supersaturating formulations. Less
Brain metastases develop in over of advanced melanoma patients and negatively impact quality of life and prognosis In a murine melanoma model we previously showed that an in situ vaccination ISV regimen combining radiation treatment and intratumoral IT injection of immunocytokine IC anti-GD antibody fused to IL along with the immune checkpoint inhibitor anti-CTLA- robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors In this study we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV anti-CTLA- B GD immunologically cold melanoma tumor ... More
Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2+, immunologically “cold”) melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50–150 mm3) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 µg daily, treatment days 6–10), and anti-CTLA-4 (100 µg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8+ and CD4+) and monocytes/macrophages (F4/80+) but no changes in FOXP3+ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (86Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic β-particle-emitting 90Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies. Less
Alginate is the structural polysaccharide of the cell wall of brown algae which is an important carbon source for marine life The depolymerization of alginate is dependent on alginate lyases Recent studies showed that the alginate utilization ability had been obtained by human gut microbes In contrast to the great number of studies on alginate lyases from marine soil organisms studies on alginate lyases from gut microbes are still limited Here the structure of a polysaccharide lyase family PL alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography which represents the cluster of two-domain PL alginate ... More
Alginate is the structural polysaccharide of the cell wall of brown algae, which is an important carbon source for marine life. The depolymerization of alginate is dependent on alginate lyases. Recent studies showed that the alginate utilization ability had been obtained by human gut microbes. In contrast to the great number of studies on alginate lyases from marine/soil organisms, studies on alginate lyases from gut microbes are still limited. Here, the structure of a polysaccharide lyase family 6 (PL6) alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography, which represents the cluster of two-domain PL6 alginate lyases from Bacteroidetes. Similar with the two-domain alginate lyase AlyGC originated from marine bacterium, both the N terminal domain (NTD) and C terminal domain (CTD) of BcAlyPL6 show right-handed parallel β-helix fold. However, unlike AlyGC, which forms a homodimer, BcAlyPL6 functions as a monomer. Biochemical analysis indicates that the substrate binding affinity is mainly contributed by the NTD while the CTD of BcAlyPL6 is involved in the formation of −1 subsite, which is essential for substrate turnover rate. Furthermore, CTD is involved in shaping a closed catalytic pocket, and deletion of it leads to increased activity towards highly polymerized substrate. Structure comparison of PL6 family alginate lyases implies that the linkers of two-domain alginate lyases might have evolutionary relationship with the N/C terminal extension of single-domain lyases. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
Polo is a Python-based graphical user interface designed to streamline viewing and analysis of images to monitor crystal growth with a specific target to enable users of the High-Throughput Crystallization Screening Center at Hauptman-Woodward Medical Research Institute HWI to efficiently inspect their crystallization experiments Polo aims to increase efficiency reducing time spent manually reviewing crystallization images and to improve the potential of identifying positive crystallization conditions Polo provides a streamlined one-click graphical interface for the Machine Recognition of Crystallization Outcomes MARCO convolutional neural network for automated image classification as well as powerful tools to view and score crystallization images to ... More
Polo is a Python-based graphical user interface designed to streamline viewing and analysis of images to monitor crystal growth, with a specific target to enable users of the High-Throughput Crystallization Screening Center at Hauptman-Woodward Medical Research Institute (HWI) to efficiently inspect their crystallization experiments. Polo aims to increase efficiency, reducing time spent manually reviewing crystallization images, and to improve the potential of identifying positive crystallization conditions. Polo provides a streamlined one-click graphical interface for the Machine Recognition of Crystallization Outcomes (MARCO) convolutional neural network for automated image classification, as well as powerful tools to view and score crystallization images, to compare crystallization conditions, and to facilitate collaborative review of crystallization screening results. Crystallization images need not have been captured at HWI to utilize Polo's basic functionality. Polo is free to use and modify for both academic and commercial use under the terms of the copyleft GNU General Public License v3.0. Less
Light-oxygen-voltage LOV domains are widespread photosensory modules that can be used in fluorescence microscopy optogenetics and controlled production of reactive oxygen species All of the currently known LOV domains have absorption maxima in the range of to nm and it is not clear whether they can be shifted significantly using mutations Here we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP Gln to asparagine aspartate glutamate histidine lysine and arginine Absorption spectra of all of the mutants are blue-shifted with the maximal shift of nm ... More
Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins. Less
DNA enzymes also known as deoxyribozymes are synthetic single-stranded DNA molecules able to catalyze chemical reactions There are two main reasons for studying deoxyribozymes their practical value in various applications and the understanding of basic properties - such as folding and catalysis - of a biopolymer that is of central importance for life Compared to ribozymes the DNA enzymes have a potential value as tools for industrial or therapeutic applications owing to more cost-effective synthesis and higher stability The first crystal structure of a deoxyribozyme demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support catalysis ... More
DNA enzymes, also known as deoxyribozymes, are synthetic single-stranded DNA molecules able
to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their
practical value in various applications, and the understanding of basic properties - such as folding
and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the
DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to
more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme
demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support
catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra,
Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving
deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although
the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These
crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear
mechanistic picture of the specific features of different RNA ligating and RNA cleaving
deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional
deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified
RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA
modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal
is to elucidate the structural basis for recognition of the methylated nucleobase by solving the
crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an
uncleavable analog of the RNA substrate, containing either methylated and unmethylated
adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the
crystallization process. Two structures for unmethylated and one of the methylated RNA substrate
analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating
deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of
the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as
cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The
structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two
RNA substrates during the crystallization process. In the future, other strategies are necessary to
solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and
RNA-ligating deoxyribozymes investigated in this study.
The second project deals with synthetic RNA aptamers that were identified by in vitro selection to
mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples
2
of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have
recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs
of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA
aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokesshifted
emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone)
derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019).
The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently
cationic sidechains that are bound by the RNA in their protonated phenol form, while emission
occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+
complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA
fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray
crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding
site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the
Chili aptamer to activate red and green-emitting chromophores. Less
to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their
practical value in various applications, and the understanding of basic properties - such as folding
and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the
DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to
more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme
demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support
catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra,
Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving
deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although
the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These
crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear
mechanistic picture of the specific features of different RNA ligating and RNA cleaving
deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional
deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified
RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA
modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal
is to elucidate the structural basis for recognition of the methylated nucleobase by solving the
crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an
uncleavable analog of the RNA substrate, containing either methylated and unmethylated
adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the
crystallization process. Two structures for unmethylated and one of the methylated RNA substrate
analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating
deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of
the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as
cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The
structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two
RNA substrates during the crystallization process. In the future, other strategies are necessary to
solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and
RNA-ligating deoxyribozymes investigated in this study.
The second project deals with synthetic RNA aptamers that were identified by in vitro selection to
mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples
2
of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have
recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs
of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA
aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokesshifted
emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone)
derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019).
The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently
cationic sidechains that are bound by the RNA in their protonated phenol form, while emission
occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+
complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA
fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray
crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding
site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the
Chili aptamer to activate red and green-emitting chromophores. Less
Unique challenges in formulating the next generation of active pharmaceutical ingredients APIs into stable formulations demand analytical tools not currently available using common benchtop methods Herein we review approaches to address some of these challenges through leveraging nonlinear optical NLO interactions between light and matter Applications in dissolution testing polymorphism and accelerated stability testing highlight the breadth of these methods The specificity of second harmonic generation SHG to chiral crystals supports rapid polymorphism analysis at the limit of individual crystals and informs formulations designs to address solubility challenges common in emerging drug candidates Coherent anti-Stokes Raman spectroscopy CARS and stimulated ... More
Unique challenges in formulating the next generation of active pharmaceutical ingredients (APIs) into stable formulations demand analytical tools not currently available using common benchtop methods. Herein, we review approaches to address some of these challenges through leveraging nonlinear optical (NLO) interactions between light and matter. Applications in dissolution testing, polymorphism, and accelerated stability testing highlight the breadth of these methods. The specificity of second harmonic generation (SHG) to chiral crystals supports rapid polymorphism analysis at the limit of individual crystals and informs formulations designs to address solubility challenges common in emerging drug candidates. Coherent anti-Stokes Raman spectroscopy (CARS) and stimulated Raman spectroscopy (SRS) provide vibration-specific microscopy of final dosage forms to inform composition at video-rate acquisition speeds and ~1 μm spatial resolution. Recent results are reviewed to illustrate challenges in designing formulations for emerging drug candidates and opportunities for the development of NLO tools tailored to meet them. Less
Nicotinamide adenine dinucleotide NAD is a key molecule in cellular bioenergetics and signalling Various bacterial pathogens release NADase enzymes into the host cell that deplete the host s NAD pool thereby causing rapid cell death Here we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa The enzymes harbour a tuberculosis necrotizing toxin TNT domain and are predominately present in pathogenic species The X-ray structure of the homodimeric A fumigatus protein reveals unique properties including N-linked glycosylation and a Ca -binding site whose occupancy regulates activity The structure ... More
Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms. Less
Understanding antibody responses to SARS-CoV- is indispensable for the development of containment measures to overcome the current COVID- pandemic Recent studies showed that serum from convalescent patients can display variable neutralization capacities Still it remains unclear whether there are specific signatures that can be used to predict neutralization Here we performed a detailed analysis of sera from a cohort of recovered healthcare workers and we addressed their SARS-CoV- antibody response by ELISA against SARS-CoV- Spike receptor binding domain and nucleoprotein Both ELISA methods detected sustained levels of serum IgG against both antigens Yet the majority of individuals from our cohort ... More
Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization. Less
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution yet the unique contributions of multimeric IgM antibodies to infection control are not well understood This is partially due to the difficulty of distinguishing low-affinity IgM secreted rapidly by plasmablasts from high-affinity antibodies derived from later-arising memory cells We developed a pipeline to express B cell receptors BCRs from Plasmodium falciparum specific IgM and IgG human memory B cells MBCs as both IgM and IgG molecules BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity Crystallization of one IgM MBC-derived antibody complexed with antigen defined a linear epitope within ... More
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines. Less
We previously developed a panel of one-step real-time quantitative reverse transcription PCR one-step qRT-PCR hereafter referred to as qRT-PCR assays to assess compound efficacy However these high-cost conventional qRT-PCR manual assays are not amenable to high-throughput screen HTS analysis in a time-sensitive and complex drug discovery process Here we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines including primary T cells This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery ... More
We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel. Less
We report the development automation and validation of a D microfluidic liver-on-a-chip for high throughput hepatotoxicity screening the OrganoPlate LiverTox The model is comprised of aggregates of induced pluripotent stem cell iPSC -derived hepatocytes iHep seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP- monoblasts differentiated to macrophages seeded in the vascular channel of the well Mimetas OrganoPlate -lane A key component of high throughput screening is automation and we report a protocol to seed dose collect and replenish media and add assay reagents in the OrganoPlate -lane using a standard laboratory liquid ... More
We report the development, automation and validation of a 3D, microfluidic liver-on-a-chip for high throughput hepatotoxicity screening, the OrganoPlate LiverTox™. The model is comprised of aggregates of induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP-1 monoblasts differentiated to macrophages seeded in the vascular channel of the 96 well Mimetas OrganoPlate 2-lane. A key component of high throughput screening is automation and we report a protocol to seed, dose, collect and replenish media and add assay reagents in the OrganoPlate 2-lane using a standard laboratory liquid handling robot. A combination of secretome measurements and image-based analysis was used to demonstrate stable 15 day cell viability, albumin and urea secretion. Over the same time-period, CYP3A4 activity increased and alpha-fetoprotein secretion decreased suggesting further maturation of the iHeps. Troglitazone, a clinical hepatotoxin, was chosen as a control compound for validation studies. Albumin, urea, hepatocyte nuclear size and viability staining provided Robust Z’factors > 0.2 in plates treated 72 h with 180 μM troglitazone compared with a vehicle control. The viability assay provided the most robust statistic for a Robust Z’ factor = 0.6. A small library of 159 compounds with known liver effects was added to the OrganoPlate LiverTox model for 72 h at 50 μM and the Toxicological Prioritization scores were calculated. A follow up dose-response evaluation of select hits revealed the albumin assay to be the most sensitive in calculating TC50 values. This platform provides a robust, novel model which can be used for high throughput hepatotoxicity screening. Less
Aiming at streamlining GPCR production from E coli inclusion bodies for structural analysis we present a generic approach to assess and optimize refolding yield through thermostability analysis Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent -Diethylamino- - -maleimidophenyl - -methylcoumarin CPM Successful expression purification and refolding is shown for two G protein-coupled receptors GPCR the sphingosine- -phosphate receptor S P and the orphan receptor GPR Refolded receptors were subjected to lipidic cubic phase crystallization screening
Here a protocol is presented to facilitate the creation of large volumes L of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs The method is based upon an understanding of the protein crystal phase diagram and how that knowledge can be utilized The method is divided into three stages optimizing crystal morphology transitioning to batch and scaling Stage involves finding well diffracting single crystals hopefully but not necessarily presenting in a cube-like morphology In Stage the Stage condition is optimized by crystal growth time This strategy can transform crystals grown by vapor diffusion to batch Once ... More
Here, a protocol is presented to facilitate the creation of large volumes (> 100 µL) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects. Less
The human neuropeptide Y NPY Y receptor Y R plays essential roles in food intake bone formation and mood regulation and has been considered an important drug target for obesity and anxiety However development of drugs targeting Y R remains challenging with no success in clinical application yet Here we report the crystal structure of Y R bound to a selective antagonist JNJ- at resolution The structure reveals molecular details of the ligand-binding mode of Y R Combined with mutagenesis studies the Y R structure provides insights into key factors that define antagonistic activity of diverse antagonists Comparison with the ... More
The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design. Less
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects Fully automated data collection is now possible at several synchrotrons which means that the number of samples tested at the synchrotron is currently increasing rapidly Therefore the protein crystallography research communities at the University of Oulu Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron In ... More
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank. Less
Mutations in two different domains of the ubiquitously expressed TRIM protein give rise to two clinically separate diseases one of which is Limb-girdle muscular dystrophy type H LGMD H Uncovering the muscle-specific role of TRIM in LGMD H pathogenesis has proven difficult as neurogenic phenotypes independent of LGMD H pathology are present in TRIM KO mice We previously established a platform to study LGMD H pathogenesis using Drosophila melanogaster as a model Here we show that LGMD H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM Furthermore transgenic expression of a subset ... More
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration. Less
The Parkin co-regulated gene protein PACRG binds at the inner junction between doublet microtubules of the axoneme a structure found in flagella and cilia PACRG binds to the adaptor protein meiosis expressed gene MEIG but how they bind to microtubules is unknown Here we report the crystal structure of human PACRG in complex with MEIG PACRG adopts a helical repeat fold with a loop that interacts with MEIG Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation ... More
The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly. Less
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects Fully automated data collection is now possible at several synchrotrons which means that the number of samples tested at the synchrotron is currently increasing rapidly Therefore the protein crystallography research communities at the University of Oulu Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron In ... More
The web-based IceBear software is a versatile tool to monitor the results of
crystallization experiments and is designed to facilitate supervisor and student
communications. It also records and tracks all relevant information from
crystallization setup to PDB deposition in protein crystallography projects. Fully
automated data collection is now possible at several synchrotrons, which means
that the number of samples tested at the synchrotron is currently increasing
rapidly. Therefore, the protein crystallography research communities at the
University of Oulu, Weizmann Institute of Science and Diamond Light Source
have joined forces to automate the uploading of sample metadata to the
synchrotron. In IceBear, each crystal selected for data collection is given a
unique sample name and a crystal page is generated. Subsequently, the metadata
required for data collection are uploaded directly to the ISPyB synchrotron
database by a shipment module, and for each sample a link to the relevant
ISPyB page is stored. IceBear allows notes to be made for each sample during
cryocooling treatment and during data collection, as well as in later steps of the
structure determination. Protocols are also available to aid the recycling of pins,
pucks and dewars when the dewar returns from the synchrotron. The IceBear
database is organized around projects, and project members can easily access
the crystallization and diffraction metadata for each sample, as well as any
additional information that has been provided via the notes. The crystal page for
each sample connects the crystallization, diffraction and structural information
by providing links to the IceBear drop-viewer page and to the ISPyB datacollection page, as well as to the structure deposited in the Protein Data Bank. Less
crystallization experiments and is designed to facilitate supervisor and student
communications. It also records and tracks all relevant information from
crystallization setup to PDB deposition in protein crystallography projects. Fully
automated data collection is now possible at several synchrotrons, which means
that the number of samples tested at the synchrotron is currently increasing
rapidly. Therefore, the protein crystallography research communities at the
University of Oulu, Weizmann Institute of Science and Diamond Light Source
have joined forces to automate the uploading of sample metadata to the
synchrotron. In IceBear, each crystal selected for data collection is given a
unique sample name and a crystal page is generated. Subsequently, the metadata
required for data collection are uploaded directly to the ISPyB synchrotron
database by a shipment module, and for each sample a link to the relevant
ISPyB page is stored. IceBear allows notes to be made for each sample during
cryocooling treatment and during data collection, as well as in later steps of the
structure determination. Protocols are also available to aid the recycling of pins,
pucks and dewars when the dewar returns from the synchrotron. The IceBear
database is organized around projects, and project members can easily access
the crystallization and diffraction metadata for each sample, as well as any
additional information that has been provided via the notes. The crystal page for
each sample connects the crystallization, diffraction and structural information
by providing links to the IceBear drop-viewer page and to the ISPyB datacollection page, as well as to the structure deposited in the Protein Data Bank. Less
The unique crystallization properties of the antenna protein C-phycocyanin C-PC from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives Remarkably the crystal dimensions vary from a few micrometres as used in serial crystallography to several hundred micrometres with a very diverse crystal morphology More than unique single-crystal X-ray diffraction data sets were collected from randomly selected crystals and analysed The addition of small-molecule additives revealed three new crystal packings of C-PC which are discussed ... More
The unique crystallization properties of the antenna protein C-phycocyanin (C-PC) from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed. C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives. Remarkably, the crystal dimensions vary from a few micrometres, as used in serial crystallography, to several hundred micrometres, with a very diverse crystal morphology. More than 100 unique single-crystal X-ray diffraction data sets were collected from randomly selected crystals and analysed. The addition of small-molecule additives revealed three new crystal packings of C-PC, which are discussed in detail. The high propensity of this protein to crystallize, combined with its natural blue colour and its fluorescence characteristics, make it an excellent candidate as a superior and highly adaptable model system in crystallography. C-PC can be used in technical and methods development approaches for X-ray and neutron diffraction techniques, and as a system for comprehending the fundamental principles of protein crystallography. Less
The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA Here we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides Kinetic NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension and in part because of the greater distance between the attacking RNA primer -hydroxyl and the phosphate of the incoming threo-nucleotide ... More
The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3′-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands. Less
Myxococcus xanthus displays two types of motilities i e Social S and Adventurous A The pole-to-pole reversals of these motility regulator proteins is the key to this process Here we determined resolution crystal structure of MglC which revealed that despite sharing sequence identity both MglB and MglC adopt Regulatory Light Chain RLC family fold Interestingly MglC is structurally unique compared to the other known RLC family proteins having - shift in the orientation of functionally important helix Using isothermal titration calorimetry and gel filtration chromatography we show that MglC binds MglB in stoichiometry with submicromolar range dissociation constant Using combination ... More
Myxococcus xanthus displays two types of motilities i.e. Social (S) and Adventurous (A). The pole-to-pole reversals of these motility regulator proteins is the key to this process. Here, we determined ~1.85 Å resolution crystal structure of MglC, which revealed that despite sharing <9% sequence identity, both MglB and MglC adopt Regulatory Light Chain 7 (RLC7) family fold. Interestingly, MglC is structurally unique compared to the other known RLC7 family proteins having ~30°-40° shift in the orientation of functionally important α2 helix. Using isothermal titration calorimetry and gel filtration chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using combination of small angle X-ray scattering and molecular docking studies, we show that MglBC complex is formed by MglC homodimer sandwiched between two homodimers of MglB. Less
Monoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges Hydrogel microsphere formulations offer a route to quicker patient-friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force Crystals of an intact monoclonal antibody are prepared as a concentrated suspension mg mL which is then encapsulated within hydrogel microspheres with diameters as small as m The hydrogel microspheres ... More
Monoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders, but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges. Hydrogel microsphere formulations offer a route to quicker, patient-friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force. Crystals of an intact monoclonal antibody are prepared as a concentrated suspension (>300 mg mL−1) which is then encapsulated within hydrogel microspheres with diameters as small as 30 µm. The hydrogel microspheres contain up to 56 wt% (dry basis) monoclonal antibody and release within 4 days under in vitro dissolution conditions. The hydrogel microspheres are concentrated into densely packed suspensions containing up to 300 mg mL−1 monoclonal antibody to evaluate their flow. These hydrogel formulations shear-thin and have lower viscosity when compared to both liquid and suspended crystal forms of the monoclonal antibody, demonstrating the potential of hydrogel microsphere encapsulants as a carrier which can mask undesirable flow properties of concentrated antibody therapeutics. Less
White spot syndrome virus WSSV the causative agent of white spot disease WSD severely affecting crustacean life forms is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target leading to various anti-cancer drugs The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase wTS Here we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate dUMP and methotrexate at a resolution of and ... More
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- and other SARS-like-CoVs encode tandem macrodomains within non-structural protein nsp The first macrodomain Mac is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose MAR from target proteins Mac likely counters host-mediated anti-viral ADP-ribosylation a posttranslational modification that is part of the host response to viral infections Mac is essential for pathogenesis in multiple animal models of CoV infection implicating it as a virulence factor and potential therapeutic target Here we report the crystal structure of SARS-CoV- Mac in complex with ADP-ribose SARS-CoV- SARS-CoV and MERS-CoV Mac exhibit similar structural folds and all proteins ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. Less
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork In eukaryotes proliferating cell nuclear antigen PCNA plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability PCNA from several organisms including Saccharomyces cerevisiae has been structurally characterised However the structural analyses of PCNA from fungal pathogens are limited Recently we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S cerevisiae Still it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic ... More
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. Less
Neurotensin receptor NTSR and related G protein coupled receptors of the ghrelin family are clinically unexploited and several mechanistic aspects of their activation and inactivation have remained unclear Enabled by a new crystallization design we present five new structures apo-state NTSR as well as complexes with nonpeptide inverse agonists SR and SR A partial agonist RTI- a and the novel full agonist SRI- providing structural rationales on how ligands modulate NTSR The inverse agonists favor a large extracellular opening of helices VI and VII undescribed so far for NTSR causing a constriction of the intracellular portion In contrast the full ... More
Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors. Less
Background Huntington's disease HD is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors Using shotgun sequencing we investigated the gut microbiome composition in the R transgenic mouse model of HD from to weeks of age early adolescent through to adult stages Targeted metabolomics was also performed on the blood plasma of these mice n per group at weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile Results ... More
Background Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors. Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors. Using shotgun sequencing, we investigated the gut microbiome composition in the R6/1 transgenic mouse model of HD from 4 to 12 weeks of age (early adolescent through to adult stages). Targeted metabolomics was also performed on the blood plasma of these mice (n = 9 per group) at 12 weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile. Results Modelled time profiles of each species, KEGG Orthologs and bacterial genes, revealed heightened volatility in the R6/1 mice, indicating potential early effects of the HD mutation in the gut. In addition to gut dysbiosis in R6/1 mice at 12 weeks of age, gut microbiome function was perturbed. In particular, the butanoate metabolism pathway was elevated, suggesting increased production of the protective SCFA, butyrate, in the gut. No significant alterations were found in the plasma butyrate and propionate levels in the R6/1 mice at 12 weeks of age. The statistical integration of the metagenomics and metabolomics unraveled several Bacteroides species that were negatively correlated with ATP and pipecolic acid in the plasma. Conclusions The present study revealed the instability of the HD gut microbiome during the pre-motor symptomatic stage of the disease which may have dire consequences on the host's health. Perturbation of the HD gut microbiome function prior to significant cognitive and motor dysfunction suggest the potential role of the gut in modulating the pathogenesis of HD, potentially via specific altered plasma metabolites which mediate gut-brain signaling. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
In this thesis the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin NmHR is presented Members of the rhodopsin family such as NmHR are integral membrane proteins comprising seven helices and a retinal chromophore which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive Through photoactivation the retinal chromophore undergoes an isomerization reaction which then drives conformational changes in the protein Through variations in residue composition rhodopsin can catalyze diverse chemical reactions including pumping protons sodium or chloride ions Chloride transport is a fundamental process in biology and crucial ... More
In this thesis, the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin (NmHR) is presented. Members of the rhodopsin family, such as NmHR, are integral membrane proteins comprising seven α helices and a retinal chromophore, which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive. Through photoactivation, the retinal chromophore undergoes an isomerization reaction, which then drives conformational changes in the protein. Through variations in residue composition, rhodopsin can catalyze diverse chemical reactions, including pumping protons, sodium, or chloride ions. Chloride transport is a fundamental process in biology and crucial for maintaining the electrochemical balance of the cell.
The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.
Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.
This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less
The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.
Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.
This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less
How have complex brains evolved from simple circuits Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei the output structures of the cerebellum Using single-nucleus RNA sequencing in mice chickens and humans as well as STARmap spatial transcriptomic analysis and whole central nervous system projection tracing we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in ... More
How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole–central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets. Less
Single-cell RNA-sequencing technologies are ideally placed to resolve intratumoral heterogeneity However the lack of coverage across key mutation hotspots has precluded the correlation of genetic and transcriptional readouts from the same single cell To overcome this we developed TARGET-seq a protocol for TARGETed high-sensitivity single-cell mutational analysis with extremely low allelic dropout rates parallel RNA SEQuencing and cell-surface proteomics Here we present a detailed step-by-step protocol for TARGET-seq including troubleshooting tips approaches for automation and methods for high-throughput multiplexing of libraries
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types Here we examine how specific transcriptomic types of mouse prefrontal cortex PFC projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks We found that most types projected to multiple targets and most targets received projections from multiple types except PFC PAG periaqueductal gray By comparing Ca activity of the molecularly homogeneous PFC PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice ... More
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics. Less
The ongoing severe acute respiratory syndrome coronavirus SARS-CoV- pandemic has devastated the global economy and claimed more than million lives presenting an urgent global health crisis To identify host factors required for infection by SARS-CoV- and seasonal coronaviruses we designed a focused high-coverage CRISPR-Cas library targeting members of a recently published SARS-CoV- protein interactome We leveraged the compact nature of this library to systematically screen SARS-CoV- at two physiologically relevant temperatures along with three related coronaviruses human coronavirus E HCoV- E HCoV-NL and HCoV-OC allowing us to probe this interactome at a much higher resolution than genome-scale studies This approach ... More
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks. Less
Electron crystallography of sub-micrometre-sized D protein crystals has emerged recently as a valuable field of structural biology In meso crystallization methods utilizing lipidic mesophases particularly lipidic cubic phases LCPs can produce high-quality D crystals of membrane proteins MPs A major step towards realizing D electron crystallography of MP crystals grown in meso is to demonstrate electron diffraction from such crystals The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals Additionally the crystals have to be thin enough to let electrons traverse them without significant multiple scattering In the present ... More
Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media. Less
Structural studies of challenging targets such as G protein-coupled receptors GPCRs have accelerated during the last several years due to the development of new approaches including small-wedge and serial crystallography Here we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase LCP grown microcrystals of two human GPCRs Cysteinyl leukotriene receptors and CysLT R and CysLT R in complex with various antagonists Five datasets were collected using small-wedge synchrotron crystallography SWSX at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions Two datasets were collected using X-ray free electron laser XFEL serial ... More
Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing. Less
In this paper we applied EfficientNet a scalable deep convolution neural network with a custom data augmentation stage to a public protein crystallization image dataset called MARCO The MARCO dataset has protein crystallization images collected from several well-known institutions In our experiments EfficientNet outperformed the accuracies reported in the previous studies and it reached an overall testing and validation accuracy on the dataset Also EfficientNet achieved crystal detection accuracy in testing data which is significant improvement over existing studies
Excitatory amino acid transporters EAATs harness Na K and H gradients for fast and efficient glutamate removal from the synaptic cleft Since each glutamate is cotransported with three Na ions Na gradients are the predominant driving force for glutamate uptake We combined all-atom molecular dynamics simulations fluorescence spectroscopy and x-ray crystallography to study Na substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type Na -only bound GltPh at - resolution revealed the fully open outward-facing state primed for subsequent substrate binding Simulations and kinetic experiments established that only the binding of two Na ... More
Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations. Less
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly Vertebrate transferrins coordinate iron through interactions with two tyrosines an aspartate a histidine and a carbonate anion and conformational changes that occur upon iron binding and release have been described Much less is known about the structure and functions of insect transferrin- Tsf which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf differs from that of the vertebrate transferrins Here we report the first crystal structure resolution of an ... More
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin-1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl-lobe adopts a novel orientation and contacts with the amino-lobe. The structure revealed coordination of a single Fe3+ ion in the amino-lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans. Less
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families Here we present a functional and structural characterization of two proteins of viral rhodopsin group OLPVR and VirChR Functional analysis of VirChR shows that it is a highly selective Na K -conducting channel and in contrast to known cation channelrhodopsins it is impermeable to Ca ions We show that upon illumination VirChR is ... More
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability. Less
The role of gene expression during learning and in short-term memories has been studied extensively but less is known about remote memories which can persist for a lifetime Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning Out of a vast plasticity-coding space we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory Unexpectedly astrocytes and ... More
The role of gene expression during learning and in short-term memories has been studied extensively1,2,3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage. Less
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase LCP However almost all previous crystallization trials have used monoacylglycerols with - cis- -octadecanoyl -rac-glycerol MO being the most widely used lipid We now report that EROCOC mixed with w w cholesterol Fig serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A A receptor A AR The structures of EROCOC -matrix grown A AR crystals were determined at resolution by serial synchrotron rotation crystallography at a cryogenic temperature and at ... More
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method. Less
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan As Lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a ... More
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan As lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes cross-linked to the matrix How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may ... More
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide–peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2,3,4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein and substrate engineering based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F A MD simulation further ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein engineering and substrate modifications based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F A MD simulation ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
- - protein isoforms regulate multiple processes in eukaryotes including apoptosis and cell division - - proteins preferentially recognize phosphorylated unstructured motifs justifying the protein-peptide binding approach to study - - phosphotarget complexes Tethering of human - - with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides simultaneously facilitating purification and crystallization Nevertheless the broader applicability to other - - isoforms and phosphopeptides was unclear Here we designed a novel - - chimera with a conserved phosphopeptide from BAD whose complex with - - is a gatekeeper of apoptosis regulation The ... More
14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the “chimeric” approach for studies of other relevant 14-3-3 complexes. Less
SARS-CoV- is a betacoronavirus virus responsible for the COVID- pandemic Here we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody CV isolated from a patient infected with SARS-CoV- in complex with the receptor binding domain RBD The structure reveals CV s epitope overlaps with the human ACE receptor binding site thus providing the structural basis for its neutralization by preventing ACE binding
PDGF VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine paracrine and endocrine mechanisms We investigated organ-specific metabolic roles of Drosophila PDGF VEGF-like factors Pvfs We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf signals to the Drosophila hepatocyte-like cells oenocytes to suppress lipid synthesis by activating the Pi K Akt TOR signaling cascade in the oenocytes Functionally this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis We find ... More
PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine, and endocrine mechanisms. We investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells. Less