1194 Citations
-adrenergic receptors ARs are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems The therapeutic potential of ARs however is largely unexploited and hampered by the scarcity of subtype-selective ligands Moreover several aminergic drugs either show off-target binding to ARs or fail to interact with the desired subtype Here we report the crystal structure of human BAR bound to the inverse agonist -cyclazosin enabled by the fusion to a DARPin crystallization chaperone The BAR structure allows the identification of two unique secondary binding pockets By structural comparison of BAR with ARs and by constructing BAR- CAR ... More
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype. Less
Alphaviruses such as Ross River virus RRV chikungunya virus CHIKV Sindbis virus SINV and Venezuelan equine encephalitis virus VEEV are mosquito-borne pathogens that can cause arthritis or encephalitis diseases Nonstructural protein nsP of alphaviruses possesses RNA-dependent RNA polymerase RdRp activity essential for viral RNA replication No D structure has been available for nsP of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs Here we report crystal structures of the RdRp domain of nsP from both RRV and SINV determined at resolutions of and The structure of the alphavirus RdRp domain appears ... More
Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication. Less
Protein homeostasis proteostasis refers to the dynamic regulation of a stable and functional proteome The extensive proteostasis network includes integrated cellular mechanisms that control biogenesis folding trafficking and degradation of proteins Posttranslational modification and protein degradation are key pathways that minimize homeostatic perturbations however disease occurs when these processes become dysregulated Loss of protein homeostasis such as by an increase in misfolded proteins contributes to the pathology of many disorders including cancers to neurodegenerative diseases Maintaining the integrity of the proteome is essential for viability but cells continuously face extracellular and intracellular stresses that destabilize protein homeostasis Collaborations among several ... More
Protein homeostasis (proteostasis) refers to the dynamic regulation of a
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. Less
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. Less
Activity-regulated cytoskeleton-associated protein Arc is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals However the mechanistic and structural basis of Arc function is little understood Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands In addition the NTD and CTD both function in Arc oligomerization including assembly of retrovirus-like capsid involved in intercellular signaling We produced and characterised six ultra-high-affinity anti-Arc nanobodies Nb The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs ... More
Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo. Less
Identification of selective deubiquitinase DUB inhibitors is critical for probe development to further understand and explore DUB biological function Here we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs In screening the compound library against multiple DUBs in parallel we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery
Deubiquitinating enzymes DUBs are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors Despite growing interest in DUB biological function and potential as therapeutic targets few selective small-molecule inhibitors and no approved drugs currently exist To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs spanning three well-characterized DUB families Promising hit compounds were validated in a series of counter-screens and orthogonal assays as well as ... More
Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes. Less
Base excision repair BER is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species BER is initiated by DNA glycosylases each of which repairs a particular class of base damage NTHL a bifunctional DNA glycosylase possesses both glycolytic and -lytic activities with a preference for oxidized pyrimidine substrates Defects in human NTHL drive a class of polyposis colorectal cancer We report the first X-ray crystal structure of hNTHL revealing an open conformation not previously observed in the bacterial orthologs In this conformation the six-helical barrel domain comprising the helix-hairpin-helix HhH DNA binding motif ... More
Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1. Less
Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations Although several antibody-based targeted approaches have been readily available for single-cell analysis so far only the mass spectrometry methodology can offer unbiased proteome profiling While this strategy has only recently emerged it has already demonstrated unparalleled analytical power quantifying proteins in single cells Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell ... More
Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells, which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations. Although several antibody-based targeted approaches have been readily available for single-cell analysis, so far only the mass spectrometry methodology can offer unbiased proteome profiling. While this strategy has only recently emerged, it has already demonstrated unparalleled analytical power quantifying >1000 proteins in single cells. Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell types and instrumentation. This chapter provides a typical example of mass spectrometry-based single-cell proteomics workflow with details about the critical steps of analysis and alternative methods useful for optimization purposes. Less
The cancer-associated fibroblast CAF marker podoplanin PDPN is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target Despite its biomedical relevance basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized thus challenging drug development Here we utilize a high throughput platform to elucidate the PDPN cell surface interactome and uncover the neutrophil protein CD as a new binding partner Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling growth and actomyosin contractility among other processes Moreover cellular assays demonstrate ... More
The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment. Less
Over the past years single-cell sequencing has become very popular For this reason many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population the choice of the best protocol can be difficult due to the high abundance of available protocols instruments and options For this reason here we describe the Smart-seq protocol for full-length mRNA sequencing of single cell This protocol can be easily optimized in every molecular ... More
Over the past 7 years, single-cell sequencing has become very popular. For this reason, many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique. For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population, the choice of the best protocol can be difficult, due to the high abundance of available protocols, instruments, and options. For this reason, here we describe the Smart-seq2 protocol for full-length mRNA sequencing of single cell. This protocol can be easily optimized in every molecular biology laboratory provided with standard laboratory equipment. The protocol is suitable for many different cell types, and the cost per cell is relatively small, allowing a good balance between costs and transcript coverage. Less
The COVID- pandemic is having a major impact on public health worldwide and there is an urgent need for the creation of an armamentarium of effective therapeutics including vaccines biologics and small-molecule therapeutics to combat SARS-CoV- and emerging variants Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is an attractive target for therapeutic intervention and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV- -specific antivirals We describe herein the results ... More
The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe–Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro. Less
Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic Natural products have long been a highly productive source to mine for new antimicrobials While it has been challenging to discover new and unique antimicrobial natural products numerous drugs have been derived from studying how natural products function as secondary metabolites Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure ... More
Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic. Natural products have long been a highly productive source to mine for new antimicrobials. While it has been challenging to discover new and unique antimicrobial natural products, numerous drugs have been derived from studying how natural products function as secondary metabolites. Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone. These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure costs. We developed a screening platform consisting of low pressure reversed-phase chromatographic separation of methanolic extracts of bacteria and fungi to generate a prefractionated natural product library. This platform is suitable for academic labs to screen for antimicrobial compounds. A large growth inhibitor screen against multiple pathogens and lab strains of microbes was conducted to assess the validity of the advantages of screening fraction libraries versus crude extract libraries and to search for potential new drug-like compounds. Hits were investigated for reproducibility, isolated, and purified. One compound was discovered in an antifungal screen which may be a novel lipopeptide. Less
Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes Target ubiquitination is achieved through the action of three separate enzymes the E ubiquitin-activating enzyme the E ubiquitin-conjugating enzyme and the E ubiquitin ligase TRIM E ligases are the largest family of RING-type E ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain one or two B-box domains B and B and a coiled-coil domain In addition most TRIMs possess a C-terminal substrate-binding domain which classifies them into one of eleven TRIM classes The ... More
Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes. Target ubiquitination is achieved through the action of three separate enzymes; the E1, ubiquitin-activating enzyme, the E2, ubiquitin-conjugating enzyme and the E3, ubiquitin ligase. TRIM E3 ligases are the largest family of RING-type E3 ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain, one or two B-box domains, B1 and B2, and a coiled-coil domain. In addition, most TRIMs possess a C-terminal substrate-binding domain, which classifies them into one of eleven TRIM classes. The PRYSPRY domain is the most common substrate-binding domain in humans and links class IV TRIMs to roles in cellular innate immunity. TRIM22 and TRIM6, are Class IV TRIMs that share high sequence identity with the well-studied HIV restriction factor, TRIM5, and have also been implicated in the anti-viral response. TRIM22 is reported to function directly as a viral restriction factor against RNA viruses such as, IAV, HCV and EMCV. While TRIM6 functions to activate the innate immune signalling pathway through activation of the immune signalling factor, IKK. Aspects of TRIM22 and TRIM6 function remain understudied, including their biochemical and biophysical properties and this is the focus of this study. The results described herein outline key differences in the self-association properties of these proteins in comparison to TRIM5. Furthermore, they highlight discrepancies between the ubiquitination profiles of TRIM22 and TRIM6 presented in the literature and the activity observed in this study. Overall, this emphasizes the need for further study of the roles of TRIM22 and TRIM6, to verify current proposed functions, as well as identify potential additional functions within the cell. Less
Immunomodulatory drugs IMiDs thalidomide lenalidomide and pomalidomide Pom bind to cereblon CRBN and trigger proteasomal degradation of neo-substrates IKZF leading to multiple myeloma MM cell apoptosis Pomalidomide Pom also binds albeit weakly to p -related protein kinase PRPK aka TP RK an understudied kinase reported to be associated with poor prognosis in MM patients Here we developed a series of IMiDs based on Pom and conducted a structure-activity relationship SAR study to identify a potent and selective PRPK binder Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN and suggested specific derivatization to improve affinity ... More
Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies. Less
Single cell proteomics is an emerging field of bioanalysis allowing one to capture proteome profiles of isolated single cells which is expected to yield additional biological information in comparison with bulk cell analysis Mass spectrometry-based methods provide unbiased analysis of detectable proteins limited only by technical parameters such as sensitivity which necessitates the development of best-practice workflows Here we describe the entire experimental design of single cell proteome analysis exemplified by cultured A lung adenocarcinoma cells treated with an anti-cancer drug methotrexate and utilizing tandem mass tag TMTpro labeling strategy for mass spectrometric data acquisition
Immunoglobulin G-based monoclonal antibodies mAbs have become a dominant class of biotherapeutics in recent decades Approved antibodies are mainly of the subclasses IgG IgG and IgG as well as their derivatives Over the decades the selection of IgG subclass has frequently been based on the needs of Fc gamma receptor engagement and effector functions for the desired mechanism of action while the effect on drug product developability has been less thoroughly characterized One of the major reasons is the lack of systematic understanding of the impact of IgG subclass on the molecular properties Several efforts have been made recently to ... More
Immunoglobulin G-based monoclonal antibodies (mAbs) have become a dominant class of biotherapeutics in recent decades. Approved antibodies are mainly of the subclasses IgG1, IgG2, and IgG4, as well as their derivatives. Over the decades, the selection of IgG subclass has frequently been based on the needs of Fc gamma receptor engagement and effector functions for the desired mechanism of action, while the effect on drug product developability has been less thoroughly characterized. One of the major reasons is the lack of systematic understanding of the impact of IgG subclass on the molecular properties. Several efforts have been made recently to compare molecular property differences among these IgG subclasses, but the conclusions from these studies are sometimes obscured by the interference from variable regions. To further establish mechanistic understandings, we conducted a systematic study by grafting three independent variable regions onto human IgG1, an IgG1 variant, IgG2, and an IgG4 variant constant domains and evaluating the impact of subclass and variable regions on their molecular properties. Structural and computational analysis revealed specific molecular features that potentially account for the differential behavior of the IgG subclasses observed experimentally. Our data indicate that IgG subclass plays a significant role on molecular properties, either through direct effects or via the interplay with the variable region, the IgG1 mAbs tend to have higher solubility than either IgG2 or IgG4 mAbs in a common pH 6 buffer matrix, and solution behavior relies heavily on the charge status of the antibody at the desirable pH. Less
Current coronavirus CoV vaccines primarily target immunodominant epitopes in the S subunit which are poorly conserved and susceptible to escape mutations thus threatening vaccine efficacy Here we use structure-guided protein engineering to remove the S subunit from the Middle East respiratory syndrome MERS -CoV spike S glycoprotein and develop stabilized stem SS antigens Vaccination with MERS SS elicits cross-reactive -CoV antibody responses and protects mice against lethal MERS-CoV challenge High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies Among them antibody IgG binds with high affinity to both MERS-CoV ... More
Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies. Less
The worldwide outbreak of coronavirus disease COVID- caused by severe acute respiratory syndrome coronavirus SARS-CoV- has become an established global pandemic Alongside vaccines antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID- Here we report the discovery and characterization of PF- an orally bioavailable SARS-CoV- main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles PF- has demonstrated oral activity in a mouse- adapted SARS-CoV- model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase ... More
The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse- adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency, in a phase I clinical trial in healthy human participants. Less
Many biological systems are composed of diverse single cells This diversity necessitates functional and molecular single-cell analysis Single-cell protein analysis has long relied on affinity reagents but emerging mass-spectrometry methods either label-free or multiplexed have enabled quantifying proteins per cell while simultaneously increasing the specificity of protein quantification Here we describe the Single Cell ProtEomics SCoPE protocol which uses an isobaric carrier to enhance peptide sequence identification Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation mPOP and their peptides labeled by isobaric mass tags TMT or TMTpro for multiplexed analysis ... More
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis. Less
Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze structurally Moreover high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation Here we present a facile and versatile platform for in meso membrane protein crystallization enabling rapid atomic structure determination at both cryogenic and room temperatures We apply this approach to human integral membrane proteins which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR integral membrane protein Finally we demonstrate an automated pipeline ... More
Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. Less
Target-based azole resistance in Candida albicans involves overexpression of the ERG gene encoding lanosterol -demethylase LDM and or the presence of single or multiple mutations in this enzyme Overexpression of Candida albicans LDM CaLDM Y H I T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole and weakly increased resistance to the longer-tailed azoles VT- itraconazole and posaconazole We have used as surrogates structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM His ScLDM His to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C albicans Darlington strain ... More
Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs. Less
Ectoine is a compatible solute found in many microorganisms adapted to survive in saline and other extreme environments Here it aids microorganisms to counter osmotic stress and protect their enzymes Ectoine exhibit many interesting properties that is potentially commercially exploitable and it is currently produced and found in several products on the market While ectoine is produced by whole cell synthesis the EctABC enzymes in the biosynthesis pathway of ectoine was currently not well described structurally or functionally Here we present structural and biochemical characterizations of ectoine synthase from two organisms Chromohalobacter salexigens DSM and Marinobacter sp CK We cloned ... More
Ectoine is a compatible solute found in many microorganisms adapted to survive in saline and other extreme environments. Here, it aids microorganisms to counter osmotic stress and protect their enzymes. Ectoine exhibit many interesting properties that is potentially commercially exploitable, and it is currently produced and found in several products on the market. While ectoine is produced by whole cell synthesis, the EctABC enzymes in the biosynthesis pathway of ectoine was currently not well described structurally or functionally. Here, we present structural and biochemical characterizations of ectoine synthase from two organisms, Chromohalobacter salexigens DSM3043 and Marinobacter sp. CK1. We cloned, expressed and expression optimized both candidates, and purified them by immobilized metal affinity chromatography and gel filtration. C. salexigens EctC (CSEctC) yielded 14-18 mg/L cell culture while Marinobacter sp. CK1 (MarEctC) yielded 0.75-1.5 mg/L culture. We then produced diffracting crystals of CSEctC and obtained a data set from which the structure of CSEctC was determined. We further obtained preliminary biochemical data relating to thermostability and activity from both candidates. The crystal structure from CSEctC shows that it is adapts a typical β-sandwich fold, consistent with earlier structural investigations of other EctC type proteins. This study provides a solid foundation for further research on EctC from our model organisms, and protocols and techniques developed herein can be further optimized to obtain more biochemical data about this interesting enzyme. Less
Heat-shock proteins of kDa Hsp s are vital for all life and are notably important in protein folding Hsp s use ATP binding and hydrolysis at a nucleotide-binding domain NBD to control the binding and release of client polypeptides at a substrate-binding domain SBD however the mechanistic basis for this allostery has been elusive Here we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp DnaK We then develop a theoretical model for allosteric equilibria among Hsp conformational states to explain the observations a restraining state Hsp R-ATP restricts ATP hydrolysis and binds peptides poorly whereas a stimulating ... More
Heat-shock proteins of 70 kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure. Less
CD is a tumor necrosis factor TNF receptor which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD Activation of the CD receptor provides a costimulatory signal to promote T cell B cell and NK cell activity to facilitate antitumor and anti-infection immunity Aberrant increased and focused expression of CD on many tumor cells renders CD an attractive therapeutic target for direct tumor killing However despite their use as drug targets to treat cancers the molecular basis and atomic details of CD and CD interaction remain elusive Here we report the crystal structure of human CD in ... More
CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction. Less
Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein PrPC a glycosyl-phosphatidylinositol GPI -anchored protein expressed on the cell surface The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein PrPSc with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis Prognoses are devastating with an average survival time of approximately one year after the onset of symptoms Despite the tremendous efforts PrP physiological function and its mechanism of conversion to PrPSc remain elusive This research focuses on Xray crystallographic fragment screening technique to map ... More
Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an "undruggable" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors. Less
Medium-chain triglycerides MCTs are an emerging choice to treat neurodegenerative disorders such as Alzheimer s disease They are triesters of glycerol and three medium-chain fatty acids such as capric C and caprylic C acids The availability of C C methyl esters C C ME from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs However there are few reports on enzymes that can efficiently hydrolyse C C ME to industrial specifications Here we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus LTL which hydrolyses C ... More
Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer’s disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8–C10 methyl esters (C8–C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8–C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8–C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8–C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1’s well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8–C10 fatty acids from methyl esters for the synthesis of MCTs. Less
Understanding the molecular mechanisms by which antibodies target and neutralize the HIV- envelope glycoprotein Env is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies NAbs Here we analyzed monoclonal antibodies mAbs isolated from non-human primates NHPs immunized with variants of a native flexibly linked NFL HIV- Env stabilized trimer derived from the tier clade C strain The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from to g ml IC Structural characterization using negative-stain EM and X-ray crystallography identified the variable region V of the NFL trimer to be the common ... More
Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies. Less
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type secretion system T SS and target leukocytes to subvert host defenses T SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T SS and drives secretion via a Brownian ratchet mechanism Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin an RTX leukotoxin essential for B pertussis colonization have been shown to target the RTX domain and prevent binding to the M integrin receptor Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform ... More
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines. Less
Certain genetic variants of severe acute respiratory syndrome coronavirus SARS-CoV- are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients SARS-CoV- genome sequences from patients in the Houston Methodist health care system diagnosed from January through May are reported here Prevalence of the B Alpha variant increased rapidly and caused to of new cases in the latter half of May Eleven B genomes had an E K replacement in spike protein a change also identified in other SARS-CoV- lineages Compared with non B -infected patients individuals with B ... More
Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non–B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. In the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex. Less
Determinants of protective immunity against severe acute respiratory syndrome coronavirus SARS-CoV- infection require the development of well-standardized reproducible antibody assays This need has led to the emergence of a variety of neutralization assays Head-to-head evaluation of different SARS-CoV- neutralization platforms could facilitate comparisons across studies and laboratories Five neutralization assays were compared using plasma samples from convalescent individuals with mild to moderate coronavirus disease COVID- four cell-based systems using either live recombinant SARS-CoV- or pseudotyped viral particles created with lentivirus LV or vesicular stomatitis virus VSV packaging and one surrogate enzyme-linked immunosorbent assay ELISA -based test that measures inhibition of ... More
Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. Less
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids but this technique presents challenges that impede scalable culture expansion experimental manipulation high-throughput screening and diagnostic applications We have developed a low-viscosity matrix LVM suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers We demonstrate the utility of LVM suspension culture for organoid and ... More
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine. Less
Henipaviruses are BSL- zoonotic pathogens responsible in humans for severe encephalitis Their V protein is a key player in the evasion of the host innate immune response We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain NTD and a -enriched C-terminal domain CTD The CTD is critical for V binding to DDB which is a cellular protein that is a component of the ubiquitin ligase E complex as well as binding to MDA and LGP which are two host sensors of viral RNA Here we serendipitously discovered that the Hendra virus V protein ... More
Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). The CTD is critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins. Less
In humans epidermal melanocytes are responsible for skin pigmentation defence against ultraviolet radiation and the deadliest common skin cancer melanoma Although there is substantial overlap in melanocyte development pathways between different model organisms species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved Here we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin capturing transcriptomes across different anatomical sites developmental age sexes and multiple skin tones We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood The transcriptional signature ... More
In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy. Less
CD is the only -transmembrane -TM spanning receptor of the immune system Its extracellular domain ECD is a cell surface marker of self that binds SIRP and inhibits macrophage phagocytosis and cancer immuno-therapy approaches in clinical trials are focused on blocking CD SIRP interaction We present the crystal structure of full length CD bound to the function-blocking antibody B H CD ECD is tethered to the TM domain via a six-residue peptide linker RVVSWF that forms an extended loop SWF loop with the fundamental role of inserting the side chains of W and F into the core of CD extracellular ... More
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention. Less
The macrophage migration inhibitory factor MIF family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism s Previously we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel Despite structural and functional similarity the MIF homolog D-dopachrome tautomerase also called MIF- has low sequence identity prompting the question of whether this dynamic regulatory network is conserved Here we establish the structural basis of an allosteric site in MIF- showing with solution NMR that dynamic communication is preserved in MIF- despite differences in the primary sequence X-ray crystallography ... More
The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily. Less
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for a significant proportion of hospital-acquired infections worldwide P aeruginosa infections are especially difficult to treat due to its numerous intrinsic and acquired resistance mechanisms Even though P aeruginosa was consequently recognised by the World Health Organization as a critical pathogen requiring urgent novel therapeutic agents the current pipeline for drug discovery has been unable to meet this pressing demand By rewiring its central metabolism P aeruginosa can thrive in diverse infection scenarios For example P aeruginosa can colonize the cystic fibrosis lung by metabolizing long- and short-chain fatty acids cholesterol and ... More
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for a significant proportion of hospital-acquired infections worldwide. P. aeruginosa infections are especially difficult to treat due to its numerous intrinsic and acquired resistance mechanisms. Even though P. aeruginosa was consequently recognised by the World Health Organization as a critical pathogen requiring urgent novel therapeutic agents, the current pipeline for drug discovery has been unable to meet this pressing demand. By rewiring its central metabolism, P. aeruginosa can thrive in diverse infection scenarios. For example, P. aeruginosa can colonize the cystic fibrosis lung by metabolizing long- and short-chain fatty acids, cholesterol and amino acids generated by degradation and fermentation of lung mucin. The metabolism of the above-mentioned nutrients requires a functional 2-methylcitrate cycle (2-MCC) to metabolize propionyl coenzyme A (PrCoA), a metabolic by-product. In addition, the 2-MCC also allows the utilization of propionate as a carbon source. As propionate and its derived catabolites, including PrCoA, are lethally toxic to cells, the 2-MCC is often viewed as a propionate detoxifying pathway. Generally, the 2-MCC is comprised of three core enzymes: 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (PrpD) and 2-methylisocitrate lyase (PrpB). The 2-MCC presents itself as a potential target for therapeutic intervention in P. aeruginosa; in addition to its role in carbon fixation, it is also involved in virulence and establishing infection. In this dissertation, core enzymes in the 2-MCC were characterized: PrpC, PrpD, and PrpB. The biochemical characterization entailed kinetic analysis of enzymes for the subsequent drug-design pipeline. Structural characterization of these three enzymes resulted in multiple crystal structures solved at near-atomic resolution. PrpC and PrpB were targeted for the development of novel antipseudomonal agents using high throughput fragment-based screening (HT-FBS) and X-ray crystallography. This was followed by fragment optimization, leading to one of the initial hits inhibiting PrpC activity in vitro, and preventing growth of P. aeruginosa in the presence of propionate. Putative mechanisms of inhibition are proposed based on the structural data. PrpD, on the other hand, was not included in the HT-FBS due to its non-essential nature in P. aeruginosa. However, NMR spectroscopy identified an unexpected PrpD substrate and product stereochemistry. The crystal structure of P. aeruginosa PrpD shed light on the likely active site and revealed the residues that are critical for activity. Together, these data provide an excellent foundation for targeting this class of enzymes and offers strategies aimed at further improving the inhibitory activity of the fragment hits. Less
Respiratory syncytial virus RSV is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood and vaccination against RSV remains a public health priority The RSV F glycoprotein is a major target of neutralizing antibodies and the prefusion stabilized form of F DS-Cav is under investigation as a vaccine antigen AM is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F PreF which spans two F protomers The quality of recognizing a trimer-specific epitope makes AM valuable for probing PreF-based immunogen conformation and ... More
Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens. Less
Toll-like receptors TLRs play an important role in the innate immune response While a lot is known about the structures of their extracellular parts many questions are still left unanswered when the structural basis of TLR activation is analyzed for the TLR intracellular domains Here we report the structure and dynamics of TLR toll-interleukin like TIR cytoplasmic domain in crystal and in solution We found that the TLR -TIR domain is capable of specific binding of Zn with nanomolar affinity Interactions with Zn are mediated by cysteine residues and and C is essential for the Zn binding Potential structures of ... More
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation. Less
Severe acute respiratory syndrome coronavirus SARS-CoV testing is one component of a multilayered mitigation strategy to enable safe in-person school attendance for the K school population However costs logistics and uncertainty about effectiveness are potential barriers to implementation We assessed early data from the Massachusetts K public school pooled SARS-CoV testing program which incorporates two novel design elements in-school pod pooling for assembling pools of dry anterior nasal swabs from to individuals and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test Ag RDT to assess the operational and analytical feasibility of this approach Over months individual swabs were ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) testing is one component of a multilayered mitigation strategy to enable safe in-person school attendance for the K–12 school population. However, costs, logistics, and uncertainty about effectiveness are potential barriers to implementation. We assessed early data from the Massachusetts K–12 public school pooled SARS-CoV2 testing program, which incorporates two novel design elements: in-school “pod pooling” for assembling pools of dry anterior nasal swabs from 5 to 10 individuals and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test (Ag RDT), to assess the operational and analytical feasibility of this approach. Over 3 months, 187,597 individual swabs were tested across 39,297 pools from 738 schools. The pool positivity rate was 0.8%; 98.2% of pools tested negative and 0.2% inconclusive, and 0.8% of pools submitted could not be tested. Of 310 positive pools, 70.6% had an N1 or N2 probe cycle threshold (CT) value of ≤30. In reflex testing (performed on specimens newly collected from members of the positive pool), 92.5% of fully deconvoluted pools with an N1 or N2 target CT of ≤30 identified a positive individual using the BinaxNOW test performed 1 to 3 days later. However, of 124 positive pools with full reflex testing data available for analysis, 32 (25.8%) of BinaxNOW pool deconvolution testing attempts did not identify a positive individual, requiring additional reflex testing. With sufficient staffing support and low pool positivity rates, pooled sample collection and reflex testing were feasible for schools. These early program findings confirm that screening for K–12 students and staff is achievable at scale with a scheme that incorporates in-school pooling, primary testing by reverse transcription-PCR (RT-PCR), and Ag RDT reflex/deconvolution testing. Less
BceF is a bacterial tyrosine kinase BY-kinase from Burkholderia cepacia a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections BY-kinases share no homology with mammalian kinases and thereby offer a means to develop novel and specific antivirulence drugs Here we report the crystal structure of the BceF kinase domain at resolution The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at ... More
BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence. Less
In bacteria trans-translation is the major quality control system for rescuing stalled ribosomes It is mediated by tmRNA a hybrid RNA with properties of both a tRNA and a mRNA and the small protein SmpB Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival it is a promising target for the development of novel antibiotics To facilitate screening of chemical libraries various reliable in vitro and in vivo systems have been created for assessing trans-translational activity However the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from ... More
In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens. Less
The novel betacoronavirus severe acute respiratory syndrome-coronavirus- SARS-CoV- causes a form of severe pneumonia disease called coronavirus disease COVID- To develop human neutralizing anti-SARS-CoV- antibodies antibody gene libraries from convalescent COVID- patients were constructed and recombinant antibody fragments scFv against the receptor-binding domain RBD of the spike protein were selected by phage display The antibody STE -C shows a subnanometer IC in a plaque-based live SARS-CoV- neutralization assay The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme hACE mice model The crystal structure of STE -C Fab in complex with ... More
The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. Less
A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility
Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing ATAC-seq The method combines up-front bulk Tn tagging of chromatin with flow cytometry to isolate single nuclei or cells Reagents required to generate sequencing libraries are added to the same well in ... More
Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support. Less
To identify approaches to target DNA repair vulnerabilities in cancer we discovered nanomolar potent selective low molecular weight MW allosteric inhibitors of the polymerase function of DNA polymerase Pol including ART ART inhibits the major Pol -mediated DNA repair process Theta-Mediated End Joining without targeting Non-Homologous End Joining In addition ART elicits DNA damage and synthetic lethality in BRCA - or BRCA -mutant tumour cells and enhances the effects of a PARP inhibitor Genetic perturbation screening revealed that defects in the BP Shieldin complex which cause PARP inhibitor resistance result in in vitro and in vivo sensitivity to small molecule ... More
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Less
The -lactamase of Mycobacterium tuberculosis BlaC is susceptible to inhibition by clavulanic acid The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli The variant that was found to confer the most inhibitor resistance K R as well as variant G N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance NMR relaxation experiments to probe structural and dynamic properties The G N mutant exists in solution in two almost equally populated conformations that exchange with a rate of ca s The conformational change ... More
The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (NMR) relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g., Ser104 and Ser130) and a short loop (positions 214 to 216). In the K234R mutant, the active-site dynamics are significantly diminished with respect to the wild-type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active-site dynamics on the millisecond time scale are not required for catalytic function. Less
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process However the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood Here integrated enzymatic assays protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel B cellulase from Bacillus licheniformis Hydrolytic efficiency substrate binding affinity cleavage patterns and the apparent processivity of bacterial BlCel B are significantly impacted by the cellulose size and its surface morphology BlCel B crystallographic structure was solved with ligands spanning - to - and to subsites ... More
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general. Less
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus Here we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs IgAs and IgGs reactive with human norovirus HuNoV genogroup I or II GI or GII We note three binding patterns and identify monoclonal antibodies mAbs that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen HBGA blocking assay The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO- ... More
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features. Less
Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase Lit Lit creates a lipoprotein that is less immunogenic possibly enabling the bacteria to gain a foothold in the host by stealth Here we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action Lit consists of four transmembrane helices with an extracellular cap Conserved residues map to ... More
Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis. Less
The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics The -lactamase BlaC of this pathogen prevents the use of -lactam antibiotics except in combination with a -lactamase inhibitor To understand if exposure to such inhibitors can easily result in resistance a BlaC evolution experiment was performed studying the evolutionary adaptability against the inhibitor sulbactam Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam The G S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for ... More
The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics. The β-lactamase BlaC of this pathogen prevents the use of β-lactam antibiotics, except in combination with a β-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam. Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam. The G132S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for sulbactam inhibition. Introduction of the side chain moiety of Ser132 causes the 104–105 peptide bond to assume the cis conformation and the side chain of Ser104 to be rotated toward the sulbactam adduct with which it forms a hydrogen bond not present in the wild-type enzyme. The gatekeeper residue Ile105 also moves. These changes in the entrance of the active site can explain the decreased affinity of G132S BlaC for both substrates and sulbactam. Our results show that BlaC can easily acquire a reduced sensitivity for sulbactam, with a single-amino acid mutation, which could hinder the use of combination therapies. Less
Eukaryotic proliferating cell nuclear antigen PCNA plays an essential role in orchestrating the assembly of the replisome complex stimulating processive DNA synthesis and recruiting other regulatory proteins during the DNA damage response PCNA and its binding partner network are relatively conserved in eukaryotes and it exhibits extraordinary structural similarity across species However despite this structural similarity the PCNA of a given species is rarely functional in heterologous systems In this report we determined the X-ray crystal structure of Neurospora crassa PCNA NcPCNA and compared its structure function relationship with other available PCNA studies to understand this cross-species incompatibility We found ... More
Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure–function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility. Less