344 Citations
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error Perovskites are an example of materials having exceptional optoelectronic properties but require improvement with respect to stability and toxicity as they approach commercialization Exploring new types of perovskites is key to achieving these goals In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials This pipeline incorporates image recognition that detects crystals via convolutional neural networks with accuracy and uses parameter exploration to predict an optimal material with experimental data With this framework I discovered a new type ... More
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error. Perovskites are an example of materials having exceptional optoelectronic properties, but require improvement with respect to stability and toxicity as they approach commercialization. Exploring new types of perovskites is key to achieving these goals. In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials. This pipeline incorporates image recognition that detects crystals via convolutional neural networks with 95% accuracy and uses parameter exploration to predict an optimal material with experimental data. With this framework, I discovered a new type of perovskite single crystal, (3-PLA)2PbCl4, that employs a new ligand, 3-PLA, offering avenues to higher efficiency and more stable devices. This work develops a framework for discovering and optimizing materials in a wide chemical space and provides the groundwork for identifying new materials that lie beyond known chemical spaces. Less
Selective activation of the -opioid receptor DOP has great potential for the treatment of chronic pain benefitting from ancillary anxiolytic and antidepressant-like effects Moreover DOP agonists show reduced adverse effects as compared to -opioid receptor MOP agonists that are in the spotlight of the current opioid crisis Here we report the first crystal structures of the DOP in an activated state in complex with two relevant and structurally diverse agonists the potent opioid agonist peptide KGCHM and the small-molecule agonist DPI- at and resolution respectively Our study identifies key determinants for agonist recognition receptor activation and DOP selectivity revealing crucial ... More
Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current “opioid crisis.” Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects. Less
The release of synthetic chemical pollutants in the environment is posing serious health risks Enzymes including oxygenases play a crucial role in xenobiotic degradation In the present study we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants Fosmid clones possessing dioxygenase activity were further sequenced and their genes were identified using bioinformatics tools Two positive fosmid clones SD and RW suggested the presence of -dihydroxybiphenyl -dioxygenase BphC-SD and catechol -dioxygenase C O-RW respectively Recombinant versions of these enzymes were purified ... More
The release of synthetic chemical pollutants in the environment is posing serious health risks. Enzymes, including oxygenases, play a crucial role in xenobiotic degradation. In the present study, we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants. Fosmid clones possessing dioxygenase activity were further sequenced, and their genes were identified using bioinformatics tools. Two positive fosmid clones, SD3 and RW1, suggested the presence of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC-SD3) and catechol 2,3-dioxygenase (C23O-RW1), respectively. Recombinant versions of these enzymes were purified to examine their pollutant-degrading abilities. The crystal structure of BphC-SD3 was determined at 2.6-Å resolution, revealing a two-domain architecture, i.e., N-terminal and C-terminal domains, with the sequential arrangement of βαβββ in each domain, characteristic of Fe-dependent class II type I extradiol dioxygenases. The structure also reveals the presence of conserved amino acids lining the catalytic pocket and Fe3+ metal ion in the large funnel-shaped active site in the C-terminal domain. Further studies suggest that Fe3+ bound in the BphC-SD3 active site probably imparts aerobic stability. We further demonstrate the potential application of BphC-SD3 in biosensing of catecholic compounds. The halotolerant and oxygen-resistant properties of these enzymes reported in this study make them potential candidates for bioremediation and biosensing applications. Less
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens SAgs which cross-link major histocompatibility complex class II MHC II molecule and T-cell receptor TCR to stimulate large numbers of T cells at extremely low concentrations SAgs are associated with food poisoning and toxic shock syndrome To date genetically distinct staphylococcal SAgs have been reported This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N SEN SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal -grasp domain Amino acid and structure alignments revealed that several critical ... More
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens (SAgs), which cross-link major histocompatibility complex class II (MHC II) molecule and T-cell receptor (TCR) to stimulate large numbers of T cells at extremely low concentrations. SAgs are associated with food poisoning and toxic shock syndrome. To date, 26 genetically distinct staphylococcal SAgs have been reported. This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N (SEN). SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal β-grasp domain. Amino acid and structure alignments revealed that several critical amino acids that are proposed to be responsible for MHC II and TCR molecule engagements are variable in SEN, suggesting that SEN may adopt a different binding mode to its cellular receptors. This work helps better understand the mechanisms of action of SAgs. Less
The elicitation of broadly neutralizing antibodies bNAbs against the HIV- envelope glycoprotein Env trimer remains a major vaccine challenge Most cross-conserved protein determinants are occluded by self-N-glycan shielding limiting B cell recognition of the underlying polypeptide surface The exceptions to the contiguous glycan shield include the conserved receptor CD binding site CD bs and glycoprotein gp elements proximal to the furin cleavage site Accordingly we performed heterologous trimer-liposome prime boosting in rabbits to drive B cells specific for cross-conserved sites To preferentially expose the CD bs to B cells we eliminated proximal N-glycans while maintaining the native-like state of the ... More
The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination Less
Well-ordered HIV- envelope glycoprotein Env trimers are prioritized for clinical evaluation and there is a need for an improved understanding about how elicited B cell responses evolve following immunization To accomplish this we prime-boosted rhesus macaques with clade C NFL trimers and identified unique Ab lineages from single-sorted Env-specific memory B cells We traced all lineages in high-throughput heavy chain HC repertoire Rep-seq data generated from multiple immune compartments and time points and expressed several as monoclonal Abs mAbs Our results revealed broad dissemination and high levels of somatic hypermutation SHM of most lineages including tier virus neutralizing lineages following ... More
Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages. Less
The investigational drugs E indisulam and tasisulam aryl-sulfonamides promote the degradation of the splicing factor RBM in a proteasome-dependent mechanism While the activity critically depends on the Cullin RING ligase substrate receptor DCAF the molecular details remain elusive Here we present the cryo-EM structure of the DDB -DCAF -DDA core ligase complex bound to RBM and E at resolution together with crystal structures of engineered subcomplexes We show that DCAF adopts a novel fold stabilized by DDA and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF Our data demonstrates how aryl-sulfonamides ... More
The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the Cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at 4.4 Å resolution, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a novel fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrates how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders. Less
The class B family of G-protein-coupled receptors GPCRs has long been a paradigm for peptide hormone recognition and signal transduction One class B GPCR the glucagon-like peptide- receptor GLP- R has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease The previously determined structures of inactive GLP- R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of designed mutations Here we systematically summarize all mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical ... More
The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins. Less
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase polyketide synthase NRPS PKS followed by a number of tailoring' steps to form the two ring systems that are present in the mature product It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels Alder reaction Here the crystal structure of a selenomethionine-labelled truncated form of IdmH IdmH- was solved using single-wavelength anomalous dispersion SAD phasing This truncated variant allows consistent and easy crystallization but importantly the structure was used as a search model in molecular replacement allowing the full-length ... More
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of `tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels–Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99–107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+β barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels–Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels–Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399. Less
Recently two groups of rhodopsin genes were identified in large double-stranded DNA viruses The structure and function of viral rhodopsins are unknown We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II OLPVRII of group It forms a pentamer with a symmetrical bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of arginines whereas phenylalanines form a hydrophobic barrier in its exit The proton donor E is placed in the helix B The structure is unique among the known rhodopsins Structural and functional data and molecular dynamics suggest that OLPVRII ... More
Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists. Less
Macrophage migration inhibitory factor MIF is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions the information obtained from these models is biased towards a specific species In experimental science results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans Syrian golden hamster Mesocricetus auratus is a clinically relevant animal model for multiple human diseases Hence the major objectives of this study were to characterize the structure and function of Mesocricetus auratus ... More
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize the structure and function of Mesocricetus auratus MIF (MaMIF) and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant MaMIF was cloned, expressed and purified in a bacterial expression system. The MaMIF primary sequence, biochemical properties, and crystal structure analysis showed greater similarity with human MIF. The crystal structure of MaMIF illustrates that it forms a homotrimer as known in human and mouse. However, MaMIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that MaMIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of MaMIF into HapT1 pancreatic tumor-bearing hamsters significantly enhanced the tumor growth and tumor-associated angiogenesis. Together, the current study shows a structural and functional similarity between the hamster and human MIF. Moreover, it has demonstrated that a high level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo. Less
Macrophage migration inhibitory factor MIF is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions the information obtained from these models is biased towards a specific species In experimental science results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans Syrian golden hamster Mesocricetus auratus is a clinically relevant animal model for multiple human diseases Hence the major objectives of this study were to characterize structure and function of hamster MIF and ... More
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize structure and function of hamster MIF, and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant hamster MIF (rha-MIF) was cloned, expressed and purified in bacterial expression system. The rha-MIF primary sequence, biochemical properties and crystal structure analysis showed a greater similarity with human MIF. The crystal structure of hamster MIF illustrates that it forms a homotrimer as known in human and mouse. However, hamster MIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that rha-MIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of rha-MIF into HapT1 pancreatic tumor bearing hamsters significantly enhanced the tumor growth and tumor associated angiogenesis. Together, the current study shows a structural and functional similarity between hamster and human MIF. Moreover, it has demonstrated that a high-level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo. Less
Rational structure-based drug design SBDD relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process While SBDD has proven successful for many drug-discovery projects its application to G protein-coupled receptors GPCRs has been limited owing to extreme difficulties with their crystallization Here a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands taking advantage of the serial femtosecond crystallography approach which obviates the need for large crystals and ... More
Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins. Less
Musashi- MSI belongs to Musashi family of RNA binding proteins RBP Like Musashi- MSI it is overexpressed in a variety of cancers and is a promising therapeutic target Both MSI proteins contain two N-terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs Previously we have identified several inhibitors of MSI all of which bind to MSI as well In order to design MSI -specific inhibitors and compare the differences of binding mode of the inhibitors we set out to solve the structure of MSI -RRM the key motif that is responsible for the binding Here we ... More
Musashi-2 (MSI2) belongs to Musashi family of RNA binding proteins (RBP). Like Musashi-1 (MSI1), it is overexpressed in a variety of cancers and is a promising therapeutic target. Both MSI proteins contain two N-terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs. Previously, we have identified several inhibitors of MSI1, all of which bind to MSI2 as well. In order to design MSI2-specific inhibitors and compare the differences of binding mode of the inhibitors, we set out to solve the structure of MSI2-RRM1, the key motif that is responsible for the binding. Here, we report the crystal structure and the first NMR solution structure of MSI2-RRM1, and compare these to the structures of MSI1-RBD1 and other RBPs. A high degree of structural similarity was observed between the crystal and solution NMR structures. MSI2-RRM1 shows a highly similar overall folding topology to MSI1-RBD1 and other RBPs. The structural information of MSI2-RRM1 will be helpful for understanding MSI2-RNA interaction and for guiding rational drug design of MSI2-specific inhibitors. Less
The G protein coupled cysteinyl leukotriene receptor CysLT R mediates inflammatory processes and plays a major role in numerous disorders including asthma allergic rhinitis cardiovascular disease and cancer Selective CysLT R antagonists are widely prescribed as antiasthmatic drugs however these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects To gain deeper understanding into the functional mechanisms of CysLTRs we determined the crystal structures of CysLT R bound to two chemically distinct antagonists zafirlukast and pranlukast The structures reveal unique ligand-binding modes and signaling mechanisms including lateral ligand access to the orthosteric pocket between transmembrane ... More
The G protein–coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs. Less
A H N virus predominated recent influenza seasons which has resulted in the rigorous investigation of haemagglutinin but whether neuraminidase NA has undergone antigenic change and contributed to the predominance of A H N virus is unknown Here we show that the NA of the circulating A H N viruses has experienced significant antigenic drift since compared with the A Hong Kong vaccine strain This antigenic drift was mainly caused by amino acid mutations at NA residues S N S T introducing an N-linked glycosylation site at residue and As a result the binding of the NA of A H ... More
A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines. Less
Human anti-HIV- broadly neutralizing antibodies bNAbs protect against infection in animal models However bNAbs have not been elicited by vaccination in diverse wild-type animals or humans in part because B cells expressing the precursors of these antibodies do not recognize most HIV- envelopes Envs Immunogens have been designed that activate these B cell precursors in vivo but they also activate competing off-target responses Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody iv that selects for naive human B cells expressing immunoglobulin light chains with amino acid complementarity determining region s a key ... More
Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5–amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)–specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1. Less
Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes
Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest In light of this there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes Here we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus HIV- broadly neutralizing antibody b iglb We determined the crystal structure of two anti-idiotypes in complex with iglb and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb Immunization ... More
Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies. Less
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another Here histidine residues are introduced into the hydrophobic core of a coiled-coil trimer generating a peptide that self-assembles upon the addition of metal ions HisAD the resulting peptide is unstructured in the absence of metal and folds selectively to form an -helical construct upon complexation with Cu II and Ni II but not Co II or Zn II The structure and metal-binding ability of HisAD is probed using a combination of circular dichroism CD spectroscopy analytical ... More
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(II) and Ni(II) but not Co(II) or Zn(II). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(II) and Ni(II) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(II) complex reveals the trimeric HisAD peptide coordinates three Cu(II) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed. Less
Toxin-antitoxin TA gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance TA loci are classified into various types based upon the characteristics of their antitoxins with those in type II expressing proteic antitoxins Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal PIN domain containing conserved amino acids considered essential for activity The vapBC virulence-associated protein TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae NTHi In this study the crystal structure of ... More
Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivo. Less
Melatonin N-acetyl- -methoxytryptamine is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the regulation of blood pressure and core body temperature oncogenesis and immune function Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from -hydroxytryptamine -HT or serotonin and modulates sleep and wakefulness by activating two high-affinity G-protein-coupled receptors type A MT and type B MT Shift work travel and ubiquitous artificial lighting can disrupt natural circadian rhythms as a result sleep disorders affect a substantial population in modern society and pose ... More
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors. Less
The human MT and MT melatonin receptors are G protein-coupled receptors GPCRs involved in the regulation of circadian rhythm and sleep patterns Drug development efforts target both receptors for treatment of insomnia circadian rhythm and mood disorders and cancer while MT has also been implicated in type diabetes T D Here we report the X-ray Free Electron Laser XFEL structures of the human MT receptor in complex with agonists -phenylmelatonin -pmt and ramelteon at resolutions of and respectively along with two structures of function-related mutants H A superscripts represent the Ballesteros-Weinstein residue numbering nomenclature and N D obtained in complex ... More
The human MT11 and MT22 melatonin receptors are G protein-coupled receptors (GPCRs) involved in the regulation of circadian rhythm and sleep patterns3. Drug development efforts target both receptors for treatment of insomnia, circadian rhythm and mood disorders, and cancer3, while MT2 has also been implicated in type 2 diabetes (T2D)4,5. Here we report the X-ray Free Electron Laser (XFEL) structures of the human MT2 receptor in complex with agonists 2-phenylmelatonin (2-pmt) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants, H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-pmt. Comparison of the MT2 structures with MT18 reveals that, despite the fact that the orthosteric ligand-binding site residues are conserved, there are notable conformational variations as well as differences in [3H]-melatonin dissociation kinetics that provide new insights into the selectivity between melatonin receptor subtypes. In addition to the membrane-buried lateral ligand entry channel that is also observed in MT1, the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data supporting a prominent role for the intramembrane ligand entry in both receptors, while simultaneously suggesting the possibility of an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents. Less
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications Recently the first sodium-pumping rhodopsin KR from Krokinobacter eikastus was discovered and characterized However the existing structures of KR are contradictory and the mechanism of Na pumping is not yet understood Here we present a structure of the cationic non H light-driven pump at physiological pH in its pentameric form We also present atomic structures and functional data on the KR and its mutants including potassium pumps which show that oligomerization of the microbial ... More
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics. Less
The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN I possesses at least putative genes encoding enzymes that belong to the -hydrolase superfamily One of those genes the metallo-hydrolase-encoding igni was cloned and heterologously expressed in Pichia pastoris The enzyme produced was purified in its catalytically active form The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of The crystal belonged to space group R with unit-cell parameters a b c It is suggested that it contains one monomer of Igni within the asymmetric unit
NEMO is an essential component in the activation of the canonical NF- B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF- B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the high-resolution structure of the unbound IKK -binding domain of NEMO that will greatly facilitate the design of NEMO IKK inhibitors The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a ... More
NEMO is an essential component in the activation of the canonical NF-?B pathway and exerts its function by recruiting the I?B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-?B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKK�-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKK�-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKK�-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes. Less
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors GPCRs Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A A adenosine receptor A AAR D N and S A Both structures present an overall active-like conformation however the variants show key changes in the activation motif NPxxY Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability ... More
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A2A adenosine receptor (A2AAR), D522.50N and S913.39A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G protein signaling. Utilizing the D2.50N variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery. Less
Many drugs target the serotonin A receptor -HT AR including second-generation antipsychotics that also target the dopamine D receptor D R These drugs often produce severe side effects due to non-selective binding to other aminergic receptors Here we report the structures of human -HT AR in complex with the second-generation antipsychotics risperidone and zotepine These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket the movements of which are important for receptor activation -HT AR is structurally similar to -HT CR but possesses a unique side-extended cavity near the ... More
Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs. Less
Nramp family transporters expressed in organisms from bacteria to humans enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport We present high-resolution structures of Deinococcus radiodurans Dra Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere Strikingly while metal transport requires cycling from outward- to inward-open states efficient proton transport still occurs in outward-locked but not inward-locked DraNramp We propose a model in which metal and proton enter the transporter via the same external pathway ... More
Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Less
Mycobacterium tuberculosis Mtb the main causative agent of tuberculosis TB is naturally resistant to -lactam antibiotics due to the production of the extended spectrum -lactamase BlaC -Lactam -lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB However still little is known of the exact mechanism of BlaC inhibition by the -lactamase inhibitors currently approved for clinical use clavulanic acid sulbactam tazobactam and avibactam Here we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors The Da adduct derived from clavulanate and the trans-enamine ... More
Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other β-lactamases are discussed. Less
Prostaglandin E receptor EP a G-protein-coupled receptor is involved in disorders such as cancer and autoimmune disease Here we report the crystal structure of human EP in complex with its antagonist ONO-AE - and an inhibitory antibody at resolution The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer proximal to the highly conserved Arg residue in the seventh transmembrane domain Functional and docking studies demonstrate that the natural agonist PGE binds in a similar manner This structural information also provides insight into the ligand ... More
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family. Less
Toxin antitoxin TA systems are involved in diverse physiological processes in prokaryotes but their exact role in Mycobacterium tuberculosis Mtb virulence and in vivo stress adaptation has not been extensively studied Here we demonstrate that the VapBC TA module is essential for Mtb to establish infection in guinea pigs RNA-sequencing revealed that overexpression of VapC toxin results in metabolic slowdown suggesting that modulation of the growth rate is an essential strategy for in vivo survival Interestingly overexpression of VapC resulted in the upregulation of chromosomal TA genes suggesting the existence of highly coordinated crosstalk among TA systems In this study ... More
Toxin–antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin–substrate and toxin–antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb. Less
Human respiratory syncytial virus HRSV is a negative-stranded RNA virus that causes a globally prevalent respiratory infection which can cause lifethreatening illness particularly in the young elderly and immunocompromised HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase RdRp For replication this complex comprises the phosphoprotein P and the large protein L whereas for transcription the M - protein is also required M - is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M - surface such that binding of these ... More
Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause lifethreatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90 – 110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences. Less
Human muscarinic receptor M is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors Muscarinic receptors are targets for multiple neurodegenerative diseases The challenge has been designing subtype selective ligands against one of the five muscarinic receptors We report high resolution structures of a thermostabilized mutant M receptor bound to a subtype selective antagonist AF-DX and a non-selective antagonist NMS The thermostabilizing mutation S R in M was predicted using a theoretical strategy previously developed in our group Comparison of the crystal structures and pharmacological properties of the M receptor shows that the ... More
Human muscarinic receptor, M2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptor compared to M3 receptor leads to subtype selectivity of AF-DX 384. Less
Kinesin- transports numerous cellular cargoes along microtubules The kinesin- light chain KLC mediates cargo binding and regulates kinesin- motility To investigate the molecular basis for kinesin- recruitment and activation by cargoes we solved the crystal structure of the KLC tetratricopeptide repeat TPR domain bound to the cargo JIP This combined with biophysical and molecular evolutionary analyses reveals a kinesin- cargo binding site located on KLC TPR which is conserved in homologs from sponges to humans In the complex JIP crosslinks two KLC TPR domains via their TPR s We show that TPR forms a dimer interface that mimics JIP binding ... More
Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms. Less
Frizzled receptors FZDs are class-F G-protein-coupled receptors GPCRs that function in Wnt signalling and are essential for developing and adult organisms As central mediators in this complex signalling pathway FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research Here we present an atomic-resolution structure of the human Frizzled receptor FZD transmembrane domain in the absence of a bound ligand The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed and is distinct from all other GPCR structures reported so far Within this ... More
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs. Less
The avian influenza A H N virus continues to cause human infections in China and is a major ongoing public health concern Five epidemic waves of A H N infection have occurred since and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves Moreover highly pathogenic avian influenza HPAI A H N viruses were also isolated during the fifth epidemic wave Here we present a detailed structural and biochemical analysis of the surface hemagglutinin HA antigen ... More
The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that, compared to the 2013 virus HAs, the fifth-wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9) HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth-wave H7 HAs analyzed in this study. Less
Angiotensin II AngII plays a central role in regulating human blood pressure which is mainly mediated by interactions between AngII and the G-protein-coupled receptors GPCRs AngII type receptor AT R and AngII type receptor AT R We have solved the crystal structure of human AT R binding the peptide ligand Sar Ile AngII and its specific antibody at - resolution Sar Ile AngII interacts with both the core binding domain where the small-molecule ligands of AT R and AT R bind and the extended binding domain which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor We ... More
Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the ‘core’ binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the ‘extended’ binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs. Less
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality However we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity which is critically required to enable rational design of degraders Here we utilize comprehensive characterization of the ligand dependent CRBN BRD interaction to demonstrate that binding between proteins that have not evolved to interact is plastic Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations which are selectively bound by ligands We demonstrate that computational protein-protein docking can ... More
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands. Less
-Galactosidases EC are retaining glycosidases that cleave terminal -linked galactose residues from glycoconjugate substrates -Galactosidases take part in the turnover of cell wall associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals Deficiency of human -galactosidase A -Gal A causes Fabry disease FD a heritable X-linked lysosomal storage disorder characterized by accumulation of globotriaosylceramide Gb and globotriaosylsphingosine lyso-Gb Current management of FD involves enzyme-replacement therapy ERT An activity-based probe ABP covalently labeling the catalytic nucleophile of -Gal A has been previously designed to study -galactosidases for use in FD therapy Here we report that this ABP ... More
α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. Less
Epstein-Barr virus EBV is a causative agent of infectious mononucleosis and is associated with new cases of cancer and deaths annually Subunit vaccines against this pathogen have focused on the gp glycoprotein and remain unsuccessful We isolated human antibodies recognizing the EBV fusion machinery gH gL and gB from rare memory B cells One anti-gH gL antibody AMMO potently neutralized infection of B cells and epithelial cells the two major cell types targeted by EBV We determined a cryo-electron microscopy reconstruction of the gH gL-gp -AMMO complex and demonstrated that AMMO bound to a discontinuous epitope formed by both gH ... More
Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells; the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden. Less
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein related lipid transfer StART domains that are implicated in intracellular non-vesicular lipid transport A new family of membrane-anchored StARkins was recently identified including six members Lam Lam in the yeast Saccharomyces cerevisiae Lam Lam are anchored to the endoplasmic reticulum ER membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast To better understand the biological roles of these proteins we carried out a structure-function analysis of the second StARkin domain of Lam here termed Lam S NMR ... More
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein–related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1–Lam6, in the yeast Saccharomyces cerevisiae. Lam1–Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling “head-first” entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress. Less
Mitochondrial Carriers MCs are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism The ADP ATP Carriers AACs are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP In human four different AACs isoforms are described which are expressed in tissue-specific manner They are involved in different genetic diseases and play a role in cancerogenesis Up to now only the structures of the bovine isoform and yeast isoforms and AAC have been determined in one particular conformation obtained in complex ... More
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded. Less
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum Pf circumsporozoite protein PfCSP from several subjects immunized with an attenuated Pf whole-sporozoite SPZ vaccine Sanaria PfSPZ Vaccine Passive transfer of one of these antibodies monoclonal antibody CIS conferred high-level sterile protection in two different mouse models of malaria infection The affinity and stoichiometry of CIS binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain The first ... More
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design. Less
Drugs frequently require interactions with multiple targets via a process known as polypharmacology to achieve their therapeutic actions Currently drugs targeting several serotonin receptors including the -HT C receptor are useful for treating obesity drug abuse and schizophrenia The competing challenges of developing selective -HT C receptor ligands or creating drugs with a defined polypharmacological profile especially aimed at G protein-coupled receptors GPCRs remain extremely difficult Here we solved two structures of the -HT C receptor in complex with the highly promiscuous agonist ergotamine and the -HT A-C receptor-selective inverse agonist ritanserin at resolutions of and respectively We analyzed their ... More
Drugs frequently require interactions with multiple targets — via a process known as polypharmacology — to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin, at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs. Less
Protein crystallography has significantly advanced in recent years with in situ data collection in which crystals are placed in the X-ray beam within their growth medium being a major point of focus In situ methods eliminate the need to harvest crystals a previously unavoidable drawback particularly for often small membrane-protein crystals Here we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature C and under cryogenic conditions The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive easy to make and compatible with automated ... More
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min. Less
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle and are thus drug targets for the treatment of insomnia Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source we determined structures of human orexin receptor in complex with the subtype-selective antagonist EMPA N-ethyl- - -methoxy-pyridin- -yl - toluene- -sulfonyl -amino -N-pyridin- -ylmethyl-acetamide at - and - resolution In comparison with the non-subtype-selective antagonist suvorexant EMPA contacted fewer residues through hydrogen bonds at the orthosteric site explaining the faster dissociation rate Comparisons among these OX R structures in complex with selective antagonists and ... More
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors. Less
A licensed vaccine for respiratory syncytial virus RSV is unavailable and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants Recently isolated antibodies C and D are substantially more potent than palivizumab and a derivative of D is in clinical trials Here we show that unlike D C preferentially neutralizes subtype A viruses The crystal structure of C bound to the RSV fusion F protein reveals that the overall binding mode of C is similar to that of D but their angles of approach are substantially different Mutagenesis and virological studies demonstrate that RSV F residue is largely ... More
A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines. Less
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism signaling and ion homeostasis About half of all drug targets are membrane proteins Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics However the pace of IMP crystallography has been relatively slow due to great challenges in crystallization Lipid cubic phase LCP has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment ... More
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism, signaling, and ion homeostasis. About half of all drug targets are membrane proteins. Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics. However, the pace of IMP crystallography has been relatively slow due to great challenges in crystallization. Lipid cubic phase (LCP) has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment. Its bilayer is composed of neutral lipids, such as monoacylglycerols, and can accommodate a substantial amount of native lipids such as phospholipids and cholesterol. Thus, the structure and composition of LCP mimic biomembranes and therefore offer a native-like environment for membrane proteins, which is favorable for functionality and crystallization. Here, the principles for LCP formation, membrane protein reconstitution, and crystallization process are described. The successful application of LCP crystallization for a wide range of membrane proteins including receptors, complexes, transporters, channels, enzymes, membrane protein insertion chaperons, and outer membrane �-barrels is summarized. General methods and protocols for this method are also described. Less
CLYBL encodes a ubiquitously expressed mitochondrial enzyme conserved across all vertebrates whose cellular activity and pathway assignment are unknown Its homozygous loss is tolerated in seemingly healthy individuals with reduced circulating B levels being the only and consistent phenotype reported to date Here by combining enzymology structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells Cells lacking CLYBL accumulate citramalyl-CoA an intermediate in the C -dicarboxylate metabolic pathway that includes itaconate a recently identified human antimicrobial metabolite and immunomodulator We report that CLYBL loss leads to a cell autonomous defect in the ... More
CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human antimicrobial metabolite and immunomodulator. We report that CLYBL loss leads to a cell autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analogue inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation. Less
Deubiquitinating enzymes DUBs have garnered significant attention as drug targets in the last years The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins especially proteins that are challenging to directly target but which are stabilized by DUB family members Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools Most reported DUB inhibitors however are polypharmacological agents possessing weak micromolar potency toward their primary target limiting their utility in target validation and mechanism studies Due to a lack of high-resolution DUB small-molecule ligand complex structures no structure-guided ... More
Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5–10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors. Less