434 Citations
The modified nucleosides -deoxy- -cyano- and -deoxy- -amido- -deazaguanosine dPreQ and dADG respectively recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster the latter of which encodes proteins that comprise the elaborate Dpd restriction modification system present in diverse bacteria Recent genetic studies implicated the dpdA dpdB and dpdC genes as encoding proteins necessary for DNA modification with dpdD dpdK contributing to the restriction phenotype Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo the elucidation of the roles of each protein ... More
The modified nucleosides 2′-deoxy-7-cyano- and 2′-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction–modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD–dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ0), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ0 into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ0-modified DNA to ADG-modified DNA. Less
G-protein metallochaperone MeaB in bacteria methylmalonic aciduria type A MMAA in humans is responsible for facilitating the delivery of adenosylcobalamin AdoCbl to methylmalonyl-CoA mutase MCM the only AdoCbl-dependent enzyme in humans Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids Here we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate GTP analog guanosine- - -methyleno triphosphate GMPPCP with the Cbl-binding domain of its target mutase enzyme MeMCMcbl This structure provides an explanation for the stimulation of the ... More
G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5′-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery. Less
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins Structural information is essential for unraveling the molecular function of proteins and structure-based drug design However there are several obstacles associated with the structural determination of proteins using X-ray crystallography such as the generation of a large amount of protein samples instability of purified proteins and difficulty in obtaining large and well-diffracting crystals all of which can prolong the process of determining the crystal structure from months to years Over the past decade new techniques and strategies have been developed to assist X-ray crystallographers in ... More
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins. Structural information is essential for unraveling the molecular function of proteins and structure-based drug design. However, there are several obstacles associated with the structural determination of proteins using X-ray crystallography, such as the generation of a large amount of protein samples, instability of purified proteins, and difficulty in obtaining large and well-diffracting crystals, all of which can prolong the process of determining the crystal structure, from months to years. Over the past decade, new techniques and strategies have been developed to assist X-ray crystallographers in overcoming some of these obstacles. In this chapter, we discuss some of these technological advances. Familiarity with these new developments would benefit researchers in both academic and industrial environments who study macromolecular structural dynamics using X-ray crystallography. Less
Voltage-gated sodium ion channels Nav are central to action potential initiation through regulating the entry of sodium ions Na into excitable cells including cardiomyocytes and neurones The -subunit of Nav consists of four homologous domains DI-DIV each consisting of six transmembrane helices S -S Helices S -S of each domain forms the lining of the central pore through which sodium ions Na enters the cell upon channel activation Helices S -S of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential Additionally Nav channels include an extracellular turret region whose role in channel ... More
Voltage-gated sodium ion channels (Nav) are central to action potential initiation through regulating the entry of sodium ions (Na+) into excitable cells including cardiomyocytes and neurones. The α-subunit of Nav consists of four homologous domains (DI-DIV), each consisting of six transmembrane helices (S1-S6). Helices S5-S6 of each domain forms the lining of the central pore through which sodium ions (Na+) enters the cell upon channel activation. Helices S1-S4 of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential. Additionally, Nav channels include an extracellular turret region, whose role in channel function is poorly understood. The C-terminal domain (CTD) of Nav, connected to the DIV-S6, interacts with various other proteins including calmodulin (CaM) and fibroblast growth factor (FGF13) and mediates various regulatory roles. The subtype Nav1.5 is primarily expressed in the heart where it initiates the cardiac action potential whereas Nav1.7 is found in the peripheral nervous system where it is associated with nociception.
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Autofluorescence-detected photothermal mid-infrared AF-PTIR microscopy was shown to enable parts-per-million detection of -indomethacin impurity in -indomethacin samples Subtle differences in the photothermal response of the UV-autofluorescence of two indomethacin crystal polymorphs were used for sub-micron chemical discrimination based on fingerprint region mid-IR spectroscopy The AF-PTIR assignment was independently confirmed by second harmonic generation SHG microscopy which was shown to reduce the total analysis time by rapidly identifying the suitable fields of view AF-PTIR microscopy has the potential to assist in the early identification of crystal form impurities in the solid dosage forms development pipeline
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers Here we show the identification and development of an affinity-enhanced T cell receptor TCR that recognizes a peptide derived from the most common KRAS mutant KRASG D presented in the context of HLA-A The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG D over KRASWT While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG D ... More
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Less
Nonribosomal peptide synthetase heterocyclization Cy domains generate biologically important oxazoline thiazoline groups found in natural products including pharmaceuticals and virulence factors such as some siderophores Cy domains catalyze consecutive condensation and cyclodehydration reactions although the mechanism is unknown To better understand Cy domain catalysis here we report the crystal structure of the second Cy domain Cy of yersiniabactin synthetase from the causative agent of the plague Yersinia pestis Our high-resolution structure of Cy adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein CP to which it is tethered We also ... More
Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. Our high-resolution structure of Cy2 adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein (CP) to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor CP that mediate donor binding. Finally, we explored domain flexibility through normal mode analysis and identified small-molecule fragment-binding sites that may inform future antibiotic design targeting Cy function. Our results suggest how CP binding may influence global Cy conformations, with consequences for active-site remodeling to facilitate the separate condensation and cyclodehydration steps as well as potential inhibitor development. Less
Chickpea is a crop that is known as a source of high-quality proteins CL-AI which belongs to the S globulin and cupin superfamily was initially identified in chickpea seeds CL-AI has recently been shown to inhibit various types of -amylases To determine its molecular mechanism the crystal structure of CL-AI was solved at a final resolution of Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association and each molecule is divided into an -chain and a -chain CL-AI has high structural similarity to other S globulins and canonical metal-dependent enzyme-related cupin proteins ... More
Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a β-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI. Less
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of D protein structures which has recently been further augmented by revolutionary advances in cryoEM However a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts limiting the overview of inherent protein physiological dynamics which play a critical role in the biological functions of proteins Here a room-temperature X-ray crystallo graphic method using temperature as a trigger to record movie-like structural snapshots has been developed The method has been used to show how TL a Da fragment undergoes ... More
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery. Less
T-cell receptor TCR immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials The safety of patients is paramount so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor ie TCR specificity and cross-reactivity We recently reported a TCR engineering campaign and selectivity profiling assay X-scan targeting a melanoma antigen gene MAGE -A peptide This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation allowing one to be advanced to T-cell immunotherapeutic clinical trials Here we present three-dimensional structural information on ... More
T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity. Less
Heavy-atom soaking has been a major method for experimental phasing but it has been difficult for membrane proteins partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding The lipid cubic phase LCP has proven to be a successful method for membrane protein crystallization but experimental phasing with LCP-grown crystals remains difficult and so far only such structures were phased experimentally Here the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion SAD phasing of crystals grown in LCP Using a single crystal the structure of the glycerol -phosphate acyltransferase ... More
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. Less
Candida Als family adhesins mediate adhesion to biological and abiotic substrates as well as fungal cell aggregation fungal-bacterial co-aggregation and biofilm formation The activity of at least two family members Als and Als is dependent on amyloid-like protein aggregation that is initiated by shear force Each Als adhesin has a -residue N-terminal Ig-like invasin region The following -residue low complexity threonine-rich T domain unfolds under shear force to expose a critical amyloid-forming segment SNGIVIVATTRTV at the interface between the Ig-like invasin domain and the T domain of Candida albicans Als Amyloid prediction programs identified six potential amyloidogenic sequences in the ... More
Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-β spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked β-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-β structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-β structures are often involved in fungal pathogenesis and potentially in antifungal therapy. Less
G protein-coupled receptors GPCRs play vital roles in human physiology and pathophysiology This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics However the production and crystallization of GPCRs for structure determination comes with many challenges In this chapter we provide a comprehensive protocol for expressing and purifying the thromboxane A receptor TPR an attractive therapeutic target for use in structure studies Guidelines for crystallizing the TPR are also included Together these procedures provide a template for generating crystal structures of the TPR and indeed other ... More
G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation recent advances in methods for data collection from multiple crystals have overcome this issue At SPring- an in situ diffraction measurement system was constructed consisting of a goniometer for a plate an articulated robot and plate storage Using this system complete data sets were obtained utilizing the small-wedge measurement method Combining this system with an ... More
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser. Less
Viruses are responsible for some of the most deadly human diseases yet available vaccines and antivirals address only a fraction of the potential viral human pathogens Here we provide a methodology for managing human herpesvirus HHV infection by covalently inactivating the HHV maturational protease via a conserved non-catalytic cysteine C Using human cytomegalovirus protease HCMV Pr as a model we screened a library of disulfides to identify molecules that tether to C and inhibit proteolysis then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs We further developed an optimized tool compound targeted ... More
Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection. Less
The receptor binding domain of spike protein of SARS-CoV- binds angitensin coverting enzyme on the surface of epithelial cells leading to fusion and entry of virus into the cell Based on our experience we described a work flow for expressing and purifying proteins and screening conditions for generating diffraction quality crystals of the complex Production and crystallization of protein complex take abut twelve days from construction of vectors to harvesting and freezing crystals for data collection
De novo-designed receptor transmembrane domains TMDs present opportunities for precise control of cellular receptor functions We developed a de novo design strategy for generating programmed membrane proteins proMPs single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor CAR that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD from monomers up to tetramers All programmed CARs stimulated substantially ... More
De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology. Less
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications Bilirubin oxidase BOD is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials BOD has been extensively investigated over the last few decades While the enzyme s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated a crystal structure of BOD from bacterial origin has never been determined Here we present the first crystal structure of BOD from Bacillus pumilus BpBOD at resolution Overall BpBOD shows high homology with the fungal enzymes however it holds a ... More
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes. Less
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents We recently developed KD a fully human antibody targeting vascular endothelial growth factor receptor VEGFR KD blocked VEGF-A and VEGF-C-mediated VEGFR activation as demonstrated by the in vitro binding and competition assays and functional cellular assays Here we report a computational model of the complex between the variable fragment of KD KD Fv and the domains and of the extracellular portion of VEGFR VEGFR D - Our modeling was guided by a priori experimental information including ... More
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents. We recently developed KD035, a fully human antibody targeting vascular endothelial growth factor receptor 2 (VEGFR2). KD035 blocked VEGF-A, and VEGF-C-mediated VEGFR2 activation, as demonstrated by the in vitro binding and competition assays and functional cellular assays. Here, we report a computational model of the complex between the variable fragment of KD035 (KD035(Fv)) and the domains 2 and 3 of the extracellular portion of VEGFR2 (VEGFR2(D2-3)). Our modeling was guided by a priori experimental information including the X-ray structures of KD035 and related antibodies, binding assays, target domain mapping and comparison of KD035 affinity for VEGFR2 from different species. The accuracy of the model was assessed by molecular dynamics simulations, and subsequently validated by mutagenesis and binding analysis. Importantly, the steps followed during the generation of this model can set a precedent for future in silico efforts aimed at the accurate description of the antibody–antigen and more broadly protein–protein complexes. Less
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold They are generally described as electron carriers that rely on a T copper center coordinated by four ligands provided by the folded polypeptide The discovery of novel cupredoxins demonstrates the high diversity of this family with variations in term of copper-binding ligands copper center geometry redox potential as well as biological function AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans AcoP presents original features highly resistant to acidic pH it possesses a constrained green-type copper center of high ... More
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network. Less
Over the last two decades fragment-based drug discovery FBDD has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods However it is also often used with great success as a primary screening technique In recent years technological advances at macromolecular crystallo graphy beamlines in terms of instrumentation beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography Here the development of ... More
Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported. Less
The human chemokines CCL and CCL bind to the G protein-coupled receptor GPCR CCR and play an important role in the trafficking of immune cells as well as cancer metastasis Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine GPCR interaction and consequently tumor metastasis Here we report the first X-ray crystal structure of a truncated CCL residues at resolution revealing molecular details crucial for protein protein interactions Although the overall structure is similar to the previously ... More
The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7–70) at 2.50 Å resolution, revealing molecular details crucial for protein–protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30’s and 40’s loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5–11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40’s loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine’s function and inhibitor development. Less
The nonpolymorphic class Ib molecule HLA-E primarily presents peptides from HLA class Ia leader peptides providing an inhibitory signal to NK cells via CD NKG interactions Although peptides of pathogenic origin can also be presented by HLA-E to T cells the molecular basis underpinning their role in antigen surveillance is largely unknown Here we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide pHLA-E from bacterial Mycobacterium tuberculosis origin and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral HIV origin The structures provided a molecular foundation to develop a novel method to introduce ... More
The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions.
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs. Less
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs. Less
Retroviral elements from endogenous retroviruses have functions in mammalian physiology The best-known examples are the envelope proteins that function in placenta development and immune suppression Porcine endogenous retroviruses PERVs are an understudied class of endogenous retroviruses that infect cultured human cells raising concern regarding porcine xenografts The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development Despite the growing interest in PERVs their envelope glycoproteins remain poorly characterized Here we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain The PERV fusion protein structure reveals a conserved class ... More
Retroviral elements from endogenous retroviruses have functions in mammalian physiology. The best-known examples are the envelope proteins that function in placenta development and immune suppression. Porcine endogenous retroviruses (PERVs) are an understudied class of endogenous retroviruses that infect cultured human cells, raising concern regarding porcine xenografts. The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development. Despite the growing interest in PERVs, their envelope glycoproteins remain poorly characterized. Here, we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain. The PERV fusion protein structure reveals a conserved class I viral fusion protein six-helix bundle. Biophysical experiments demonstrated that the thermodynamic stability of the PERV fusion protein secondary structure was the same at physiological and acidic pHs. A conserved surface analysis highlights the high degree of sequence conservation among retroviral fusogens in the chain reversal region that facilitates the large-scale conformational change required for membrane fusion. Further structural alignment of class I viral fusogens revealed a phylogenetic clustering that shows evolution into various lineages that correlate with virus mechanisms of cell entry. Our work indicates that structural dendrograms can be used to qualitatively infer insights into the fusion mechanisms of newly discovered class I viral fusogen structures. Less
-adrenergic receptors ARs are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems The therapeutic potential of ARs however is largely unexploited and hampered by the scarcity of subtype-selective ligands Moreover several aminergic drugs either show off-target binding to ARs or fail to interact with the desired subtype Here we report the crystal structure of human BAR bound to the inverse agonist -cyclazosin enabled by the fusion to a DARPin crystallization chaperone The BAR structure allows the identification of two unique secondary binding pockets By structural comparison of BAR with ARs and by constructing BAR- CAR ... More
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype. Less
Activity-regulated cytoskeleton-associated protein Arc is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals However the mechanistic and structural basis of Arc function is little understood Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands In addition the NTD and CTD both function in Arc oligomerization including assembly of retrovirus-like capsid involved in intercellular signaling We produced and characterised six ultra-high-affinity anti-Arc nanobodies Nb The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs ... More
Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo. Less
Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze structurally Moreover high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation Here we present a facile and versatile platform for in meso membrane protein crystallization enabling rapid atomic structure determination at both cryogenic and room temperatures We apply this approach to human integral membrane proteins which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR integral membrane protein Finally we demonstrate an automated pipeline ... More
Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. Less
Target-based azole resistance in Candida albicans involves overexpression of the ERG gene encoding lanosterol -demethylase LDM and or the presence of single or multiple mutations in this enzyme Overexpression of Candida albicans LDM CaLDM Y H I T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole and weakly increased resistance to the longer-tailed azoles VT- itraconazole and posaconazole We have used as surrogates structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM His ScLDM His to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C albicans Darlington strain ... More
Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs. Less
Heat-shock proteins of kDa Hsp s are vital for all life and are notably important in protein folding Hsp s use ATP binding and hydrolysis at a nucleotide-binding domain NBD to control the binding and release of client polypeptides at a substrate-binding domain SBD however the mechanistic basis for this allostery has been elusive Here we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp DnaK We then develop a theoretical model for allosteric equilibria among Hsp conformational states to explain the observations a restraining state Hsp R-ATP restricts ATP hydrolysis and binds peptides poorly whereas a stimulating ... More
Heat-shock proteins of 70 kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure. Less
Henipaviruses are BSL- zoonotic pathogens responsible in humans for severe encephalitis Their V protein is a key player in the evasion of the host innate immune response We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain NTD and a -enriched C-terminal domain CTD The CTD is critical for V binding to DDB which is a cellular protein that is a component of the ubiquitin ligase E complex as well as binding to MDA and LGP which are two host sensors of viral RNA Here we serendipitously discovered that the Hendra virus V protein ... More
Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). The CTD is critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins. Less
CD is the only -transmembrane -TM spanning receptor of the immune system Its extracellular domain ECD is a cell surface marker of self that binds SIRP and inhibits macrophage phagocytosis and cancer immuno-therapy approaches in clinical trials are focused on blocking CD SIRP interaction We present the crystal structure of full length CD bound to the function-blocking antibody B H CD ECD is tethered to the TM domain via a six-residue peptide linker RVVSWF that forms an extended loop SWF loop with the fundamental role of inserting the side chains of W and F into the core of CD extracellular ... More
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention. Less
Respiratory syncytial virus RSV is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood and vaccination against RSV remains a public health priority The RSV F glycoprotein is a major target of neutralizing antibodies and the prefusion stabilized form of F DS-Cav is under investigation as a vaccine antigen AM is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F PreF which spans two F protomers The quality of recognizing a trimer-specific epitope makes AM valuable for probing PreF-based immunogen conformation and ... More
Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens. Less
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process However the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood Here integrated enzymatic assays protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel B cellulase from Bacillus licheniformis Hydrolytic efficiency substrate binding affinity cleavage patterns and the apparent processivity of bacterial BlCel B are significantly impacted by the cellulose size and its surface morphology BlCel B crystallographic structure was solved with ligands spanning - to - and to subsites ... More
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general. Less
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus Here we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs IgAs and IgGs reactive with human norovirus HuNoV genogroup I or II GI or GII We note three binding patterns and identify monoclonal antibodies mAbs that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen HBGA blocking assay The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO- ... More
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features. Less
Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase Lit Lit creates a lipoprotein that is less immunogenic possibly enabling the bacteria to gain a foothold in the host by stealth Here we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action Lit consists of four transmembrane helices with an extracellular cap Conserved residues map to ... More
Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis. Less
The chemokine receptor CCR plays a vital role in immune surveillance and inflammation However molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive Here we report three cryo-electron microscopy structures of Gi protein-coupled CCR in a ligand-free state and in complex with the chemokine MIP- or RANTES as well as the crystal structure of MIP- -bound CCR These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR Together with functional data the structures demonstrate that chemokine-induced rearrangement of toggle switch and ... More
The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of Gi1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation. Less
Odorant-binding proteins OBPs as they occur in insects form a distinct class of proteins that apparently has no closely related representatives in other animals However ticks mites spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs In this work we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor a major pest of honey bee Varroa OBPs present six cysteines paired into three disulphide bridges but with positions in the sequence and connections different from those of their insect counterparts VdesOBP structure was determined in two closely related crystal forms ... More
Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites. Less
Plant pathogens cause disease through secreted effector proteins which act to modulate host physiology and promote infection Typically the sequences of effectors provide little functional information and further targeted experimentation is required Here we utilised a structure function approach to study SnTox an effector from the necrotrophic fungal pathogen Parastagonospora nodorum which causes cell death in wheat-lines carrying the sensitivity gene Snn We developed a workflow for the production of SnTox in a heterologous host that enabled crystal structure determination We show this approach can be successfully applied to effectors from other pathogenic fungi Complementing this an in-silico study uncovered ... More
Plant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi. The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici. We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence. Less
The design of peptides that assemble in membranes to form functional ion channels is challenging Specifically hydrophobic interactions must be designed between the peptides and at the peptide lipid interfaces simultaneously Here we take a multi-step approach towards this problem First we use rational de novo design to generate water-soluble -helical barrels with polar interiors and confirm their structures using high-resolution X-ray crystallography These -helical barrels have water-filled lumens like those of transmembrane channels Next we modify the sequences to facilitate their insertion into lipid bilayers Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse cation-selective ... More
The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide. Less
An effective HIV- vaccine will likely need to elicit broadly neutralizing antibodies bNAbs Broad and potent VRC -class bNAbs have been isolated from multiple infected individuals suggesting that they could be reproducibly elicited by vaccination Several HIV- envelope-derived germline-targeting immunogens have been designed to engage naive VRC -class precursor B cells However they also present off-target epitopes that could hinder development of VRC -class bNAbs We characterize a panel of anti-idiotypic monoclonal antibodies ai-mAbs raised against inferred-germline iGL VRC -class antibodies By leveraging binding structural and B cell sorting data we engineered a bispecific molecule derived from two ai-mAbs one ... More
An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo. Less
Antibodies are crucial to immune protection against SARS-CoV- with some in emergency use as therapeutics Here we identify human monoclonal antibodies mAbs recognizing the virus spike and focus mainly on that bind the receptor binding domain RBD We devise a competition data-driven method to map RBD binding sites We find that although antibody binding sites are widely dispersed neutralizing antibody binding is focused with nearly all highly inhibitory mAbs IC mg mL blocking receptor interaction except for one that binds a unique epitope in the N-terminal domain Many of these neutralizing mAbs use public Vgenes and are close to germline ... More
Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 mg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public Vgenes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models. Less
A bispecific antibody BsAb targeting the epidermal growth factor receptor EGFR and mesenchymal epithelial transition factor MET pathways represents a novel approach to overcome resistance to targeted therapies in patients with non small cell lung cancer In this study we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule The BsAbs were derived from different EGFR and MET parental monoclonal antibodies Initially molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET Hits were identified and further screened based on their potential to induce ... More
A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling. Less
Nicotinamide adenine dinucleotide NAD is a key molecule in cellular bioenergetics and signalling Various bacterial pathogens release NADase enzymes into the host cell that deplete the host s NAD pool thereby causing rapid cell death Here we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa The enzymes harbour a tuberculosis necrotizing toxin TNT domain and are predominately present in pathogenic species The X-ray structure of the homodimeric A fumigatus protein reveals unique properties including N-linked glycosylation and a Ca -binding site whose occupancy regulates activity The structure ... More
Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms. Less
Aiming at streamlining GPCR production from E coli inclusion bodies for structural analysis we present a generic approach to assess and optimize refolding yield through thermostability analysis Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent -Diethylamino- - -maleimidophenyl - -methylcoumarin CPM Successful expression purification and refolding is shown for two G protein-coupled receptors GPCR the sphingosine- -phosphate receptor S P and the orphan receptor GPR Refolded receptors were subjected to lipidic cubic phase crystallization screening
The human neuropeptide Y NPY Y receptor Y R plays essential roles in food intake bone formation and mood regulation and has been considered an important drug target for obesity and anxiety However development of drugs targeting Y R remains challenging with no success in clinical application yet Here we report the crystal structure of Y R bound to a selective antagonist JNJ- at resolution The structure reveals molecular details of the ligand-binding mode of Y R Combined with mutagenesis studies the Y R structure provides insights into key factors that define antagonistic activity of diverse antagonists Comparison with the ... More
The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design. Less
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects Fully automated data collection is now possible at several synchrotrons which means that the number of samples tested at the synchrotron is currently increasing rapidly Therefore the protein crystallography research communities at the University of Oulu Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron In ... More
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank. Less
Myxococcus xanthus displays two types of motilities i e Social S and Adventurous A The pole-to-pole reversals of these motility regulator proteins is the key to this process Here we determined resolution crystal structure of MglC which revealed that despite sharing sequence identity both MglB and MglC adopt Regulatory Light Chain RLC family fold Interestingly MglC is structurally unique compared to the other known RLC family proteins having - shift in the orientation of functionally important helix Using isothermal titration calorimetry and gel filtration chromatography we show that MglC binds MglB in stoichiometry with submicromolar range dissociation constant Using combination ... More
Myxococcus xanthus displays two types of motilities i.e. Social (S) and Adventurous (A). The pole-to-pole reversals of these motility regulator proteins is the key to this process. Here, we determined ~1.85 Å resolution crystal structure of MglC, which revealed that despite sharing <9% sequence identity, both MglB and MglC adopt Regulatory Light Chain 7 (RLC7) family fold. Interestingly, MglC is structurally unique compared to the other known RLC7 family proteins having ~30°-40° shift in the orientation of functionally important α2 helix. Using isothermal titration calorimetry and gel filtration chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using combination of small angle X-ray scattering and molecular docking studies, we show that MglBC complex is formed by MglC homodimer sandwiched between two homodimers of MglB. Less
Neurotensin receptor NTSR and related G protein coupled receptors of the ghrelin family are clinically unexploited and several mechanistic aspects of their activation and inactivation have remained unclear Enabled by a new crystallization design we present five new structures apo-state NTSR as well as complexes with nonpeptide inverse agonists SR and SR A partial agonist RTI- a and the novel full agonist SRI- providing structural rationales on how ligands modulate NTSR The inverse agonists favor a large extracellular opening of helices VI and VII undescribed so far for NTSR causing a constriction of the intracellular portion In contrast the full ... More
Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors. Less
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase LCP However almost all previous crystallization trials have used monoacylglycerols with - cis- -octadecanoyl -rac-glycerol MO being the most widely used lipid We now report that EROCOC mixed with w w cholesterol Fig serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A A receptor A AR The structures of EROCOC -matrix grown A AR crystals were determined at resolution by serial synchrotron rotation crystallography at a cryogenic temperature and at ... More
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein engineering and substrate modifications based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F A MD simulation ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less