1181 Citations
Human leukocyte antigen HLA alleles have been implicated as risk factors for immune-mediated adverse drug reactions The authors recently reported a strong association between HLA-A and vancomycin-induced drug reaction with eosinophilia and systemic symptoms Identification of individuals with the risk allele before or shortly after the initiation of vancomycin therapy is of great clinical importance to prevent morbidity and mortality and improve drug safety and antibiotic treatment options A prerequisite to the success of pharmacogenetic screening tests is the development of simple robust cost-effective single HLA allele test that can be implemented in routine diagnostic laboratories In this study the ... More
Human leukocyte antigen (HLA) alleles have been implicated as risk factors for immune-mediated adverse drug reactions. The authors recently reported a strong association between HLA-A*32:01 and vancomycin-induced drug reaction with eosinophilia and systemic symptoms. Identification of individuals with the risk allele before or shortly after the initiation of vancomycin therapy is of great clinical importance to prevent morbidity and mortality, and improve drug safety and antibiotic treatment options. A prerequisite to the success of pharmacogenetic screening tests is the development of simple, robust, cost-effective single HLA allele test that can be implemented in routine diagnostic laboratories. In this study, the authors developed a simple, real-time allele-specific PCR for typing the HLA-A*32:01 allele. Four-hundred and fifty-eight DNA samples including 30 HLA-A*32:01–positive samples were typed by allele-specific PCR. Compared with American Society for Histocompatibility and Immunogenetics–accredited, sequence-based, high-resolution, full-allelic HLA typing, this assay demonstrates 100% accuracy, 100% sensitivity (95% CI, 88.43% to 100%), and 100% specificity (95% CI, 99.14% to 100%). The lowest limit of detection of this assay using PowerUp SYBR Green is 10 ng of template DNA. The assay demonstrates a sensitivity and specificity to differentiate the HLA-A*32:01 allele from closely related non–HLA-A*32 alleles and may be used in clinical settings to identify individuals with the risk allele before or during the course of vancomycin therapy. Less
Yersinia pestis the causative agent of bubonic plague is one of the most lethal pathogens in recorded human history Today the concern is the possible misuse of Y pestis as an agent in bioweapons and bioterrorism Current therapies for the treatment of plague include the use of a small number of antibiotics but clinical cases of antibiotic resistance have been reported in some areas of the world Therefore the discovery of new drugs is required to combat potential Y pestis infection Here the crystal structure of the Y pestis UDP-glucose pyrophosphorylase UGP a metabolic enzyme implicated in the survival of ... More
Yersinia pestis, the causative agent of bubonic plague, is one of the most lethal pathogens in recorded human history. Today, the concern is the possible misuse of Y. pestis as an agent in bioweapons and bioterrorism. Current therapies for the treatment of plague include the use of a small number of antibiotics, but clinical cases of antibiotic resistance have been reported in some areas of the world. Therefore, the discovery of new drugs is required to combat potential Y. pestis infection. Here, the crystal structure of the Y. pestis UDP-glucose pyrophosphorylase (UGP), a metabolic enzyme implicated in the survival of Y. pestis in mouse macrophages, is described at 2.17 Å resolution. The structure provides a foundation that may enable the rational design of inhibitors and open new avenues for the development of antiplague therapeutics. Less
Second harmonic generation SHG microscopy and Raman microscopy were used for qualitative and quantitative analysis of pharmaceutical materials Prototype instruments and algorithms for sampling strategies and data analyses were developed to achieve pharmaceutical materials analysis with low limits of detection and short measurement times Manufacturing an amorphous solid dispersion ASD in which an amorphous active pharmaceutical ingredient API within polymer matrix is an effective approach to improve the solubility and bioavailability of a drug However since ASDs are generally metastable materials they can often transform to produce crystalline API with higher thermodynamic stability Analytical methods with low limits of detection ... More
Second harmonic generation (SHG) microscopy and Raman microscopy were used for
qualitative and quantitative analysis of pharmaceutical materials. Prototype instruments and
algorithms for sampling strategies and data analyses were developed to achieve pharmaceutical
materials analysis with low limits of detection and short measurement times.
Manufacturing an amorphous solid dispersion (ASD), in which an amorphous active
pharmaceutical ingredient (API) within polymer matrix, is an effective approach to improve the
solubility and bioavailability of a drug. However, since ASDs are generally metastable materials,
they can often transform to produce crystalline API with higher thermodynamic stability.
Analytical methods with low limits of detection for crystalline APIs were used to assess the
stability of ASDs. With high selectivity to noncentrosymmetric crystals, SHG microscopy was
demonstrated as an analytical tool, which exhibited a limit of detection of 10 ppm for ritonavir
Form II crystals. SHG microscopy was employed for accelerated stability testing of ASDs, which
provided a four-decade dynamic range of crystallinity for kinetic modeling. An established model
was validated by investigating nucleation and crystal growth based on SHG images. To achieve in
situ accelerated stability testing, controlled environment for in situ stability testing (CEiST) was
designed and built to provide elevated temperature and humidity, which is compatible with a
commercial SHG microscope based on our research prototype. The combination of CEiST and
SHG microscopy enabled assessment of individual crystal growth rates by single-particle tracking
and nucleation rates for individual fields of view with low Poisson noise. In addition, SHG
microscopy coupled with CEiST enabled the study of heterogeneity of crystallization kinetics
within pharmaceutical materials.
Polymorphism of APIs plays an important role in drug formulation development. Different
polymorphs of identical APIs may exhibit different physiochemical properties, e.g., solubility,
stability, and bioavailability, due to their crystal structures. Moreover, polymorph transitions may take place during the manufacturing process and storage. Therefore, analytical methods with high
speed for polymorph characterization, which can provide real-time feedback for the polymorphic
transition, have broad applications in pharmaceutical materials characterization. Raman
spectroscopy is able to determine the API polymorphism, but is hampered by the long
measurement times. In this study, two analytical methods with high speed were developed to
characterize API polymorphs. One is SHG microscopy-guided Raman spectroscopy, which
achieved the speed of 10 ms/particle for clopidogrel bisulfate. Initial classification of two different
polymorphs was based on SHG images, followed acquisition of Raman spectroscopy at the
selected positions to determine the API crystal form. Another approach is implementing of
dynamic sampling into confocal Raman microscopy to accelerate Raman image acquisition for 6-
folds. Instead of raster scanning, dynamic sampling algorithm enabled acquiring Raman spectra at
the most informative locations. The reconstructed Raman image of pharmaceutical materials has
<0.5% loss of image quality with 15.8% sampling rate. Less
qualitative and quantitative analysis of pharmaceutical materials. Prototype instruments and
algorithms for sampling strategies and data analyses were developed to achieve pharmaceutical
materials analysis with low limits of detection and short measurement times.
Manufacturing an amorphous solid dispersion (ASD), in which an amorphous active
pharmaceutical ingredient (API) within polymer matrix, is an effective approach to improve the
solubility and bioavailability of a drug. However, since ASDs are generally metastable materials,
they can often transform to produce crystalline API with higher thermodynamic stability.
Analytical methods with low limits of detection for crystalline APIs were used to assess the
stability of ASDs. With high selectivity to noncentrosymmetric crystals, SHG microscopy was
demonstrated as an analytical tool, which exhibited a limit of detection of 10 ppm for ritonavir
Form II crystals. SHG microscopy was employed for accelerated stability testing of ASDs, which
provided a four-decade dynamic range of crystallinity for kinetic modeling. An established model
was validated by investigating nucleation and crystal growth based on SHG images. To achieve in
situ accelerated stability testing, controlled environment for in situ stability testing (CEiST) was
designed and built to provide elevated temperature and humidity, which is compatible with a
commercial SHG microscope based on our research prototype. The combination of CEiST and
SHG microscopy enabled assessment of individual crystal growth rates by single-particle tracking
and nucleation rates for individual fields of view with low Poisson noise. In addition, SHG
microscopy coupled with CEiST enabled the study of heterogeneity of crystallization kinetics
within pharmaceutical materials.
Polymorphism of APIs plays an important role in drug formulation development. Different
polymorphs of identical APIs may exhibit different physiochemical properties, e.g., solubility,
stability, and bioavailability, due to their crystal structures. Moreover, polymorph transitions may take place during the manufacturing process and storage. Therefore, analytical methods with high
speed for polymorph characterization, which can provide real-time feedback for the polymorphic
transition, have broad applications in pharmaceutical materials characterization. Raman
spectroscopy is able to determine the API polymorphism, but is hampered by the long
measurement times. In this study, two analytical methods with high speed were developed to
characterize API polymorphs. One is SHG microscopy-guided Raman spectroscopy, which
achieved the speed of 10 ms/particle for clopidogrel bisulfate. Initial classification of two different
polymorphs was based on SHG images, followed acquisition of Raman spectroscopy at the
selected positions to determine the API crystal form. Another approach is implementing of
dynamic sampling into confocal Raman microscopy to accelerate Raman image acquisition for 6-
folds. Instead of raster scanning, dynamic sampling algorithm enabled acquiring Raman spectra at
the most informative locations. The reconstructed Raman image of pharmaceutical materials has
<0.5% loss of image quality with 15.8% sampling rate. Less
Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation thereby promoting bacterial resistance to environmental stressors Here we present crystal structures of amyloid-forming segments from the major curli subunit CsgA revealing steric zipper fibrils of tightly mated -sheets demonstrating a structural link between curli and human pathological amyloids D-enantiomeric peptides originally developed to interfere with Alzheimer s disease-associated amyloid- inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium Moreover as previously shown CsgA fibrils cross-seeded fibrillation of amyloid- providing support for the proposed structural resemblance and potential for cross-species amyloid interactions The presented ... More
Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated β-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer’s disease-associated amyloid-β, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-β, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases. Less
Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields Purple acid phosphatases PAPs are ubiquitous metalloenzymes which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides While some plant PAPs display a preference for ATP as the substrate others are efficient in hydrolyzing phytate or -phosphoenolpyruvate PEP PAP from red kidney bean rkbPAP is an efficient ATP- and ADPase but has no activity towards phytate The crystal structure of this enzyme in complex with an ATP analogue to resolution provides insight into the amino acid residues that play ... More
Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 Å resolution) provides insight into the amino acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production. Less
The CC chemokine receptor CCR balances immunity and tolerance by homeostatic trafficking of immune cells In cancer CCR -mediated trafficking leads to lymph node metastasis suggesting the receptor as a promising therapeutic target Here we present the crystal structure of human CCR fused to the protein Sialidase NanA by using data up to resolution The structure shows the ligand Cmp bound to an intracellular allosteric binding pocket A sulfonamide group characteristic for various chemokine receptor ligands binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix and helix We demonstrate how structural data can ... More
The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer. Less
Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse with excitatory types being less layer-restricted than expected Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types Despite this general conservation we also ... More
Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain. Less
Compositions devices methods and systems are provided for differential functionalization of a surface of a structure to support biopolymer synthesis Provided herein are processes which include use of lamps lasers and or microcontact printing to add functional groups to surfaces for the efficient and uniform synthesis of oligonucleic acids
A H N virus predominated recent influenza seasons which has resulted in the rigorous investigation of haemagglutinin but whether neuraminidase NA has undergone antigenic change and contributed to the predominance of A H N virus is unknown Here we show that the NA of the circulating A H N viruses has experienced significant antigenic drift since compared with the A Hong Kong vaccine strain This antigenic drift was mainly caused by amino acid mutations at NA residues S N S T introducing an N-linked glycosylation site at residue and As a result the binding of the NA of A H ... More
A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines. Less
We previously generated rotavirus-specific RV-specific recombinant monoclonal antibodies mAbs derived from B cells isolated from human intestinal resections Twenty-four of these mAbs were specific for the VP fragment of RV VP and most of were non-neutralizing when tested in the conventional MA cell based assay We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT- cells Most of of the non-neutralizing VP mAbs efficiently neutralized human RV in HT- cells or enteroids Serum RV neutralization titers in adults and infants were significantly higher in HT- than MA cells and adsorption ... More
We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell–based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells, including ileal enteroids and HT-29 cells. Most (18 of 20) of the “non-neutralizing” VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer. Less
Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses To this end we have adapted the viral tagging approach which bypasses the need for culture-based methods to identify host phage pairings Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells which are then individually sorted as host phage pairs followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus es We demonstrate single-cell viral tagging ... More
Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses. One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses. To this end, we have adapted the viral tagging approach, which bypasses the need for culture-based methods to identify host–phage pairings. Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells, which are then individually sorted as host–phage pairs, followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus(es). We demonstrate single-cell viral tagging using the faecal microbiome, including cross-tagging of viruses and bacteria between human subjects. A total of 363 unique host–phage pairings were predicted, most of which were subject-specific and involved previously uncharacterized viruses despite the majority of their bacterial hosts having known taxonomy. One-fifth of these pairs were confirmed by multiple individual tagged cells. Viruses targeting more than one bacterial species were conspicuously absent in the host–phage network, suggesting that phages are not major vectors of inter-species horizontal gene transfer in the human gut. A high level of cross-reactivity between phages and bacteria from different subjects was noted despite subject-specific viral profiles, which has implications for faecal microbiota transplant therapy. Less
Image-based protein phase diagram analysis is key for understanding and exploiting protein phase behavior in the biopharmaceutical field However required data analysis has become a notorious time-consuming task since high-throughput screening approaches were implemented A variety of computational tools have been developed to support analysis but these tools primarily use end point visible light images This study investigates the combined effect of end point and time-dependent image features obtained from cross-polarized and ultraviolet light features supplementary to visible light on protein phase diagram image classification In addition external validation was performed to evaluate the classification algorithm s applicability to support ... More
Image-based protein phase diagram analysis is key for understanding and exploiting protein phase behavior in the biopharmaceutical field. However, required data analysis has become a notorious time-consuming task since high-throughput screening approaches were implemented. A variety of computational tools have been developed to support analysis, but these tools primarily use end point visible light images. This study investigates the combined effect of end point and time-dependent image features obtained from cross-polarized and ultraviolet light features, supplementary to visible light, on protein phase diagram image classification. In addition, external validation was performed to evaluate the classification algorithm’s applicability to support protein phase diagram scoring. The predicted protein phase behavior classes were subsequently used to automatically construct multidimensional protein phase diagrams to prevent image information loss without complicating the used image classification algorithm. Combining end point and time-dependent features from 3 light sources resulted in a balanced accuracy of 86.4 ± 4.3%, which is comparable to or better than more complex classifiers reported in literature. External validation resulted in a correct formulation classification rate of 91.7%. Subsequent automated construction of the multidimensional protein phase diagrams, using predicted classes, allowed visualization of details such as crystallization rate and protein phase behavior type coexistence. Less
Human anti-HIV- broadly neutralizing antibodies bNAbs protect against infection in animal models However bNAbs have not been elicited by vaccination in diverse wild-type animals or humans in part because B cells expressing the precursors of these antibodies do not recognize most HIV- envelopes Envs Immunogens have been designed that activate these B cell precursors in vivo but they also activate competing off-target responses Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody iv that selects for naive human B cells expressing immunoglobulin light chains with amino acid complementarity determining region s a key ... More
Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5–amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)–specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1. Less
Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes
Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest In light of this there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes Here we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus HIV- broadly neutralizing antibody b iglb We determined the crystal structure of two anti-idiotypes in complex with iglb and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb Immunization ... More
Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies. Less
RNase H dependent PCR-enabled T-cell receptor sequencing rhTCRseq can be used to determine paired alpha beta T-cell receptor TCR clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples With the enhanced specificity of RNase H dependent PCR rhPCR it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step For single cells the protocol includes sorting of single cells into plates generation of cDNA libraries a TCR-specific amplification step a second PCR on pooled sample to generate a sequencing library and sequencing In the bulk method sorting and cDNA library ... More
RNase H–dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H–dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2–4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d. Less
Balanced fusion and fission are key for the proper function and physiology of mitochondria Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance Mgm in fungi or the related protein optic atrophy OPA in animals Mgm is required for the preservation of mitochondrial DNA in yeast whereas mutations in the OPA gene in humans are a common cause of autosomal dominant optic atrophy a genetic disorder that affects the optic nerve Mgm and OPA are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space ... More
Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3,4,5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy—a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane—not the inner membrane—fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane. Less
The -oxopurine phosphoribosyltransferases PRTs are drug targets for the treatment of parasitic diseases This is due to the fact that parasites are auxotrophic for the -oxopurine bases relying on salvage enzymes for the synthesis of their -oxopurine nucleoside monophosphates In Trypanosoma brucei the parasite that is the aetiological agent for sleeping sickness there are three -oxopurine PRT isoforms Two are specific for hypoxanthine and guanine whilst the third characterized here uses all three naturally occurring bases with similar efficiency Here we have determined crystal structures for TbrHGXPRT in complex with GMP XMP and IMP to investigate the structural basis for ... More
The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. Less
Discovery of novel Mnk inhibitors using mutation-based induced-fit virtual high-throughput screening
Mnk kinases Mnk and are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor E Engagement of the Mnk pathway is critical in acute myeloid leukemia AML leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo suggesting that targeting Mnk kinases may provide a novel approach for treating AML Here we report the development and application of a mutation-based induced-fit in silico screen to identify novel Mnk inhibitors The Mnk structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk crystal structure while carrying out ... More
Mnk kinases (Mnk1 and 2) are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor 4E. Engagement of the Mnk pathway is critical in acute myeloid leukemia (AML) leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo, suggesting that targeting Mnk kinases may provide a novel approach for treating AML. Here, we report the development and application of a mutation-based induced-fit in silico screen to identify novel Mnk inhibitors. The Mnk1 structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk1 crystal structure while carrying out docking simulations of known inhibitors. The hit compounds display activity in Mnk biochemical and cellular assays, including acute myeloid leukemia progenitors. This approach will enable further rational structure-based drug design of new Mnk inhibitors and potentially novel ways of therapeutically targeting this kinase. Less
Objective To optimize post-differentiation freeze thaw and culture conditions for induced pluripotent stem cell-derived brain microvascular endothelial cell iPSC-BMEC maturation functionality and reproducibility Methods Human iPSCs were spontaneously differentiated into BMECs protocol based on Lippmann et al Nat Biotech Dissociation and sub-culture conditions were investigated using Design of Experiments DoE an unbiased method based on structured statistical analysis of variance ANOVA DoE efficiently identifies interactions amongst experimental variables and predicts responses Our design focused on maximizing trans-endothelial electrical resistance TEER We incorporated a Mantis liquid handler to execute precise culture conditions in -well transwell systems and measured TEER over time ... More
Objective: To optimize post-differentiation freeze, thaw, and culture conditions for induced pluripotent stem cell-derived brain microvascular endothelial cell (iPSC-BMEC) maturation, functionality, and reproducibility. Methods: Human iPSCs were spontaneously differentiated into BMECs (protocol based on Lippmann et al. Nat Biotech 2012). Dissociation and sub-culture conditions were investigated using Design of Experiments (DoE), an unbiased method based on structured statistical analysis of variance (ANOVA). DoE efficiently identifies interactions amongst experimental variables and predicts responses. Our design focused on maximizing trans-endothelial electrical resistance (TEER). We incorporated a Mantis liquid handler to execute precise culture conditions in 96-well transwell systems and measured TEER over time. Top hit conditions were validated on multiple batches of iPSC-BMECs, these included post-thaw cell viability, hourly TEER reads, and immunocytochemistry (ICC). Results: We have identified a robust cryopreservation method, time course and medium formulation for sub-culturing spontaneously differentiated iPSC-BMEC. The optimized cryopreservation protocol yields high cell viability recovery allowing the ability to produce bulk batches of iPSC-BMECs, thereby minimizing interexperimental variability. Surprisingly, we found that the removal of retinoic acid and the extension to at least 7 days in culture reproducibly resulted in prolonged high TEER, approximately 500 ohms * cm2 greater than TEER peak at 48 h, in iPSC-BMEC mono-culture transwells. Our sub-culture method generates iPSC-BMECs that express the endothelial surface marker PECAM1, relevant blood–brain barrier (BBB) tight junction proteins (claudin-5, ZO-1, occludin), and transporters enriched on brain microvessels (Glut-1, transferrin receptor, insulin receptor). Conclusion: We demonstrate the power of applying DoE to fine-tune complex culture conditions to maximize cell performance. Our cryopreservation and sub-culture protocols robustly produce functional endothelial cells of the BBB that are suited to address basic cerebral vascular biology questions, study the vascular phenotype of neurological disorders, and enable high-throughput screening for drug discovery. Less
The structure of BgaR a transcriptional regulator of the lactose operon in Clostridium perfringens has been solved by SAD phasing using a mercury derivative BgaR is an exquisite sensor of lactose with a binding affinity in the low-micromolar range This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form and was compared with AraC another saccharide-binding transcriptional regulator It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold but that part of the saccharide is exposed to bulk solvent This differs from the classical ... More
The structure of BgaR, a transcriptional regulator of the lactose operon in�Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC. Less
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another Here histidine residues are introduced into the hydrophobic core of a coiled-coil trimer generating a peptide that self-assembles upon the addition of metal ions HisAD the resulting peptide is unstructured in the absence of metal and folds selectively to form an -helical construct upon complexation with Cu II and Ni II but not Co II or Zn II The structure and metal-binding ability of HisAD is probed using a combination of circular dichroism CD spectroscopy analytical ... More
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(II) and Ni(II) but not Co(II) or Zn(II). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(II) and Ni(II) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(II) complex reveals the trimeric HisAD peptide coordinates three Cu(II) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed. Less
To enable rapid selection of traits in marker-assisted breeding markers must be technically simple low-cost high-throughput and randomly distributed in a genome We developed such a technology designated as Multiplex Restriction Amplicon Sequencing MRASeq which reduces genome complexity by polymerase chain reaction PCR amplification of amplicons flanked by restriction sites The first PCR primers contain restriction site sequences at -ends preceded by - bases of specific or degenerate nucleotide sequences and then by a unique M -tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing ... More
To enable rapid selection of traits in marker-assisted breeding, markers must be technically simple, low-cost, high-throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’-ends, preceded by 6-10 bases of specific or degenerate nucleotide sequences and then by a unique M13-tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next-generation sequencing-based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species. Less
DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications Here we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase TdT in kinetically controlled conditions Information is stored in transitions between non-identical nucleotides of DNA strands To produce strands representing user-defined content nucleotide substrates are added iteratively yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation With this scheme we synthesize DNA strands carrying bits including addressing and demonstrate retrieval with streaming nanopore sequencing We ... More
DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA. Less
Transient receptor potential TRP channels are polymodal sensory transducers that respond to chemicals temperature mechanical stress and membrane voltage and are involved in vision taste olfaction hearing touch thermal perception and nociception TRP channels are implicated in numerous devastating diseases including various forms of cancer and represent important drug targets The large sizes low expression levels and conformational dynamics of TRP channels make them challenging targets for structural biology Here we present the methodology used in structural studies of TRPV a TRP channel that is highly selective for calcium and mediates Ca uptake in epithelial tissues We provide a protocol ... More
Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca2+ uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family. Less
Human NimA-related kinases Neks have multiple mitotic and non-mitotic functions but few substrates are known We systematically determined the phosphorylation-site motifs for the entire Nek kinase family except for Nek While all Nek kinases strongly select for hydrophobic residues in the position the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor and preference for basic or acidic residues in other positions Unlike Nek -Nek Nek is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine and its activity is enhanced by tyrosine auto-phosphorylation Nek dual-specificity depends on residues ... More
Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the −3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling. Less
The universally conserved N -threonylcarbamoyladenosine t A modification of tRNA is essential for translational fidelity In bacteria t A biosynthesis starts with the TsaC TsaC -catalyzed synthesis of the intermediate threonylcarbamoyl adenylate TC AMP followed by transfer of the threonylcarbamoyl TC moiety to adenine- of tRNA by the TC-transfer complex comprised of TsaB TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t A cycle We report a - crystal structure of the T maritima TC-transfer complex TmTsaB D E bound to Mg -ATP in the ATPase site and substrate analog carboxy-AMP in the TC-transfer ... More
The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system. Less
Toxin-antitoxin TA gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance TA loci are classified into various types based upon the characteristics of their antitoxins with those in type II expressing proteic antitoxins Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal PIN domain containing conserved amino acids considered essential for activity The vapBC virulence-associated protein TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae NTHi In this study the crystal structure of ... More
Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivo. Less
The HLA-A -restricted decapeptide EAAGIGILTV derived from melanoma antigen recognized by T-cells- MART- protein represents one of the best-studied tumor associated T-cell epitopes but clinical results targeting this peptide have been disappointing This limitation may reflect the dominance of the nonapeptide AAGIGILTV at the melanoma cell surface The decapeptide and nonapeptide are presented in distinct conformations by HLA-A and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly Here we studied the MEL TCR that potently recognizes the nonapeptide The structure of the MEL -HLA-A -AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide MHC ... More
The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells. Less
The human protein tyrosine phosphatase non-receptor type PTPN is a PDZ PSD- Dlg ZO- domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers Interestingly the high-risk genital human papillomavirus HPV types and target the PDZ domain of PTPN The presence of a PDZ binding motif PBM on E confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential Here we report the molecular basis of interaction between the PDZ domain of PTPN and the PBM of the HPV E protein We combined biophysical NMR and X-ray experiments to ... More
The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a PDZ (PSD-95/Dlg/ZO-1) domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers. Interestingly, the high-risk genital human papillomavirus (HPV) types 16 and 18 target the PDZ domain of PTPN3. The presence of a PDZ binding motif (PBM) on E6 confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential. Here, we report the molecular basis of interaction between the PDZ domain of PTPN3 and the PBM of the HPV E6 protein. We combined biophysical, NMR and X-ray experiments to investigate the structural and functional properties of the PDZ domain of PTPN3. We showed that the C-terminal sequences from viral proteins encompassing a PBM interact with PTPN3-PDZ with similar affinities to the endogenous PTPN3 ligand MAP kinase p38γ. PBM binding stabilizes the PDZ domain of PTPN3. We solved the X-ray structure of the PDZ domain of PTPN3 in complex with the PBM of the HPV E6 protein. The crystal structure and the NMR chemical shift mapping of the PTPN3-PDZ/peptide complex allowed us to pinpoint the main structural determinants of recognition of the C-terminal sequence of the E6 protein and the long-range perturbations induced upon PBM binding. Less
An aged circulatory environment can activate microglia reduce neural precursor cell activity and impair cognition in mice We hypothesized that brain endothelial cells BECs mediate at least some of these effects We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule VCAM a protein that facilitates vascular immune cell interactions Concomitantly levels of the shed soluble form of VCAM are prominently increased in the plasma of aged humans and mice and their plasma is sufficient to increase VCAM expression in cultured BECs and the hippocampi of young mice ... More
An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular–immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood–brain barrier as a possible target to treat age-related neurodegeneration. Less
The polymerase of negative-stranded RNA viruses consists of the large protein L and the phosphoprotein P the latter serving both as a chaperon and a cofactor for L We mapped within measles virus MeV P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain MD of P is required for gene expression MeV MD is kinked as a result of the presence of a stammer Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay By contrast P activity is rather tolerant of substitutions ... More
The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the “a” or “d” hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness. Less
Melatonin N-acetyl- -methoxytryptamine is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the regulation of blood pressure and core body temperature oncogenesis and immune function Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from -hydroxytryptamine -HT or serotonin and modulates sleep and wakefulness by activating two high-affinity G-protein-coupled receptors type A MT and type B MT Shift work travel and ubiquitous artificial lighting can disrupt natural circadian rhythms as a result sleep disorders affect a substantial population in modern society and pose ... More
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors. Less
The three members of the endocrine fibroblast growth factor FGF family designated FGF FGF and FGF mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor FGFR bound to either -Klotho or -Klotho receptors Structural analyses of ligandoccupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF and FGF to -Klotho or -Klotho respectively They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling Here we describe the crystal structure the C-terminal tail of ... More
The three members of the endocrine fibroblast growth factor (FGF) family designated FGF19, FGF21, and FGF23 mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor (FGFR) bound to either α-Klotho or β-Klotho receptors. Structural analyses of ligandoccupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF21 and FGF23 to β-Klotho or α-Klotho, respectively. They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling. Here we describe the crystal structure the C-terminal tail of FGF19 (FGF19CT) bound to sKLB and demonstrate that FGF19CT and FGF21CT bind to the same binding site on sKLB, via a multiturn D-P motif to site 1 and via a SP-S motif to the pseudoglycoside hydrolase region (site 2). Binding affinities to sKLB and cellular stimulatory activities of FGF19CT, FGF21CT, and a variety of chimeric mutants to cells expressing β-Klotho together with FGFR1c or FGFR4 were also analyzed. These experiments as well as detailed comparison of the structures of free and ligand-occupied sKLB to the structure of ligandoccupied sKLA reveal a general mechanism for recognition of endocrine FGFs by Klotho proteins and regulatory interactions with FGFRs that control their pleiotropic cellular responses. Less
The human MT and MT melatonin receptors are G protein-coupled receptors GPCRs involved in the regulation of circadian rhythm and sleep patterns Drug development efforts target both receptors for treatment of insomnia circadian rhythm and mood disorders and cancer while MT has also been implicated in type diabetes T D Here we report the X-ray Free Electron Laser XFEL structures of the human MT receptor in complex with agonists -phenylmelatonin -pmt and ramelteon at resolutions of and respectively along with two structures of function-related mutants H A superscripts represent the Ballesteros-Weinstein residue numbering nomenclature and N D obtained in complex ... More
The human MT11 and MT22 melatonin receptors are G protein-coupled receptors (GPCRs) involved in the regulation of circadian rhythm and sleep patterns3. Drug development efforts target both receptors for treatment of insomnia, circadian rhythm and mood disorders, and cancer3, while MT2 has also been implicated in type 2 diabetes (T2D)4,5. Here we report the X-ray Free Electron Laser (XFEL) structures of the human MT2 receptor in complex with agonists 2-phenylmelatonin (2-pmt) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants, H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-pmt. Comparison of the MT2 structures with MT18 reveals that, despite the fact that the orthosteric ligand-binding site residues are conserved, there are notable conformational variations as well as differences in [3H]-melatonin dissociation kinetics that provide new insights into the selectivity between melatonin receptor subtypes. In addition to the membrane-buried lateral ligand entry channel that is also observed in MT1, the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data supporting a prominent role for the intramembrane ligand entry in both receptors, while simultaneously suggesting the possibility of an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents. Less
Neurological diseases such as Alzheimer s disease AD Parkinson s disease PD Epilepsy and Multiple Sclerosis are included in the Global burden of disease study as these disorders have a high impact on public health Lack of effective treatment has motivated the researchers to perform early diagnostics by identifying new gene mutations which can improve the therapies The aim of this thesis was a genetic analysis of PD using next-generation sequencing data In this thesis whole genome sequencing WGS and whole exome sequencing WES using DNA from familial PD patients and healthy individuals was performed in order to identify the ... More
Neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Epilepsy and Multiple Sclerosis are included in the Global burden of disease study as these disorders have a high impact on public health. Lack of effective treatment has motivated the researchers to perform early diagnostics, by identifying new gene mutations, which can improve the therapies. The aim of this thesis was a genetic analysis of PD using next-generation sequencing data. In this thesis, whole genome sequencing (WGS) and whole exome sequencing (WES) using DNA from familial PD patients and healthy individuals was performed in order to identify the PD causal genes. A large repository of sporadic PD WES data and a genotyping array was used to replicate our findings. The PD patients from Germany were stratified for clinical trials on the basis of mitochondrial endo-phenotype by performing risk profiling of associated Single Nucleotide Polymorphisms (SNPs) using exome genotyping array. The sporadic PD WES and genotyping array data from International Parkinson’s disease Genomics Consortium was used to perform association tests, to determine the burden of rare variants in candidate genes of interest. Furthermore, mRNA sequencing of all the genes under the PD GWAS loci after knockdown with short hairpin RNAs was performed, to identify the actual genes contributing to PD risk and the novel pathways involved in PD. Finally, an epistatic interaction of a Mendelian PD gene and associated locus was performed to understand the joint contribution to PD risk. Taking everything into account, we identified pathogenic variants in known and some novel genes causing PD in families. On the basis of risk profiling some of the German PD patients will undergo clinical trials with coenzyme Q10 and vitamin K2. The association tests using sporadic PD data helped to identify some novel genes significantly associated with PD risk. The knockdown experiments facilitated the identification of genes contributing to PD risk in some of the PD GWAS loci. Less
The three members of the endocrine fibroblast growth factor FGF family designated FGF FGF and FGF mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor FGFR bound to either -Klotho or -Klotho receptors Structural analyses of ligand-occupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF and FGF to -Klotho or -Klotho respectively They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling Here we describe the crystal structure the C-terminal tail of ... More
The three members of the endocrine fibroblast growth factor (FGF) family designated FGF19, FGF21, and FGF23 mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor (FGFR) bound to either α-Klotho or β-Klotho receptors. Structural analyses of ligand-occupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF21 and FGF23 to β-Klotho or α-Klotho, respectively. They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling. Here we describe the crystal structure the C-terminal tail of FGF19 (FGF19CT) bound to sKLB and demonstrate that FGF19CT and FGF21CT bind to the same binding site on sKLB, via a multiturn D-P motif to site 1 and via a S-P-S motif to the pseudoglycoside hydrolase region (site 2). Binding affinities to sKLB and cellular stimulatory activities of FGF19CT, FGF21CT, and a variety of chimeric mutants to cells expressing β-Klotho together with FGFR1c or FGFR4 were also analyzed. These experiments as well as detailed comparison of the structures of free and ligand-occupied sKLB to the structure of ligand-occupied sKLA reveal a general mechanism for recognition of endocrine FGFs by Klotho proteins and regulatory interactions with FGFRs that control their pleiotropic cellular responses. Less
Many long-standing image processing problems in applied science domains are finding solutions through the application of deep learning approaches to image processing Here we present one such application the case of classifying images of protein crystallisation droplets The Collaborative Crystallisation Centre in Melbourne Australia is a medium throughput service facility that produces between five and twenty thousand images per day This submission outlines a reliable and robust machine learning pipeline that autonomously classifies these images using CSIRO s high-performance computing facilities Our pipeline achieves improved accuracies over existing implementations and delivers these results in real time We discuss the specific ... More
Many long-standing image processing problems in applied science domains are finding solutions through the application of deep learning approaches to image processing. Here we present one such application; the case of classifying images of protein crystallisation droplets. The Collaborative Crystallisation Centre in Melbourne, Australia is a medium throughput service facility that produces between five and twenty thousand images per day. This submission outlines a reliable and robust machine learning pipeline that autonomously classifies these images using CSIRO’s high-performance computing facilities. Our pipeline achieves improved accuracies over existing implementations and delivers these results in real time. We discuss the specific tools and techniques used to construct the pipeline, as well as the methodologies for testing and validating externally developed classification models. Less
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications Recently the first sodium-pumping rhodopsin KR from Krokinobacter eikastus was discovered and characterized However the existing structures of KR are contradictory and the mechanism of Na pumping is not yet understood Here we present a structure of the cationic non H light-driven pump at physiological pH in its pentameric form We also present atomic structures and functional data on the KR and its mutants including potassium pumps which show that oligomerization of the microbial ... More
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics. Less
Phox homology PX domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides markers of organelle identity in the endocytic system Although many PX domains bind the canonical endosome-enriched lipid PtdIns P others interact with alternative phosphoinositides and a precise understanding of how these specificities arise has remained elusive Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays biolayer interferometry and isothermal titration calorimetry These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns P non-specifically to various di- and tri-phosphorylated phosphoinositides bind both PtdIns ... More
Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family. Less
The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN I possesses at least putative genes encoding enzymes that belong to the -hydrolase superfamily One of those genes the metallo-hydrolase-encoding igni was cloned and heterologously expressed in Pichia pastoris The enzyme produced was purified in its catalytically active form The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of The crystal belonged to space group R with unit-cell parameters a b c It is suggested that it contains one monomer of Igni within the asymmetric unit
Inosine- -monophosphate dehydrogenase IMPDH is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents Our recent work has revealed the crucial role of one of the two structural domains i e Bateman domain in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs Thus we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH These inhibitors were shown to counteract the activation by the natural positive effector MgATP and to block ... More
Inosine-5‘-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now. Less
Poor solubility is a common challenge encountered during the development of high concentration monoclonal antibody mAb formulations but there are currently no methods that can provide predictive information on high-concentration behavior of mAbs in early discovery We explored the utility of methodologies used for determining extrapolated solubility as a way to rank-order mAbs based on their relative solubility properties We devised two approaches to accomplish this vapor diffusion technique utilized in traditional protein crystallization practice and polyethylene glycol PEG -induced precipitation and quantitation by turbidity Using a variety of in-house mAbs with known high-concentration behavior we demonstrated that both approaches ... More
Poor solubility is a common challenge encountered during the development of high concentration monoclonal antibody (mAb) formulations, but there are currently no methods that can provide predictive information on high-concentration behavior of mAbs in early discovery. We explored the utility of methodologies used for determining extrapolated solubility as a way to rank-order mAbs based on their relative solubility properties. We devised two approaches to accomplish this: 1) vapor diffusion technique utilized in traditional protein crystallization practice, and 2) polyethylene glycol (PEG)-induced precipitation and quantitation by turbidity. Using a variety of in-house mAbs with known high-concentration behavior, we demonstrated that both approaches exhibited reliable predictability of the relative solubility properties of these mAbs. Optimizing the latter approach, we developed a format that is capable of screening a large panel of mAbs in multiple pH and buffer conditions. This simple, material-saving, high-throughput approach enables the selection of superior molecules and optimal formulation conditions much earlier in the antibody discovery process, prior to time-consuming and material intensive high-concentration studies. Less
HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles This enzyme provides access to chiral -substituted alcohols that serve as building blocks in the pharmaceutical industry Wild-type HheG suffers from low thermostability which poses a significant drawback for potential applications In an attempt to thermostabilize HheG by protein engineering several single mutants at position were identified which displayed up to C increased apparent melting temperatures and up to three-fold higher activity Aromatic amino acids at position resulted even in a slightly higher enantioselectivity Crystal structures ... More
HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G. Less
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates While the impact of localization and recruitment on kinase substrate targeting can be readily assessed evaluating the relative importance of direct phosphorylation site interactions remains challenging In this study we examine the STE family of protein serine threonine kinases to investigate basic mechanisms of substrate targeting We used peptide arrays to define the phosphorylation site specificity for the majority of STE kinases and categorized them into four distinct groups Using structure-guided mutagenesis we identified key specificity-determining residues within the kinase catalytic cleft ... More
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems. Less
NEMO is an essential component in the activation of the canonical NF- B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF- B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the high-resolution structure of the unbound IKK -binding domain of NEMO that will greatly facilitate the design of NEMO IKK inhibitors The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a ... More
NEMO is an essential component in the activation of the canonical NF-?B pathway and exerts its function by recruiting the I?B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-?B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKK�-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKK�-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKK�-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes. Less
Suppressor of copper sensitivity protein C from Proteus mirabilis PmScsC is a homotrimeric disulfide isomerase that plays a role in copper tolerance which is a key virulence trait of this uropathogen Each protomer of the enzyme has an N-terminal trimerization stem residues containing a flexible linker residues connected to a thioredoxin-fold-containing catalytic domain residues Here two PmScsC variants PmScsC N and PmScsC Linker are characterized PmScsC N is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed generating a protein with dithiol oxidase rather than disulfide isomerase activity The crystal structure of PmScsC N ... More
Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance, which is a key virulence trait of this uropathogen. Each protomer of the enzyme has an N-terminal trimerization stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, two PmScsC variants, PmScsC?N and PmScsC?Linker, are characterized. PmScsC?N is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsC?N reported here reveals, as expected, a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant, PmScsC?Linker, was designed to remove the 11-amino-acid linker, and it is shown that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsC?Linker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small-angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsC?Linker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function. Less
Upon triggering by their inducer signal transduction ATPases with numerous domains STANDs initially in monomeric resting forms multimerize into large hubs that activate target macromolecules This process requires conversion of the STAND conserved core the NOD from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize In the absence of inducer autoinhibitory interactions maintain the NOD closed In particular in resting STAND proteins with an LRR- or WD -type sensor domain the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms Here we solved the first crystal structure of a ... More
Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains. Less
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors GPCRs Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A A adenosine receptor A AAR D N and S A Both structures present an overall active-like conformation however the variants show key changes in the activation motif NPxxY Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability ... More
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A2A adenosine receptor (A2AAR), D522.50N and S913.39A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G protein signaling. Utilizing the D2.50N variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery. Less
Nramp family transporters expressed in organisms from bacteria to humans enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport We present high-resolution structures of Deinococcus radiodurans Dra Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere Strikingly while metal transport requires cycling from outward- to inward-open states efficient proton transport still occurs in outward-locked but not inward-locked DraNramp We propose a model in which metal and proton enter the transporter via the same external pathway ... More
Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Less
Many drugs target the serotonin A receptor -HT AR including second-generation antipsychotics that also target the dopamine D receptor D R These drugs often produce severe side effects due to non-selective binding to other aminergic receptors Here we report the structures of human -HT AR in complex with the second-generation antipsychotics risperidone and zotepine These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket the movements of which are important for receptor activation -HT AR is structurally similar to -HT CR but possesses a unique side-extended cavity near the ... More
Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs. Less