901 Citations
Monoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges Hydrogel microsphere formulations offer a route to quicker patient-friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force Crystals of an intact monoclonal antibody are prepared as a concentrated suspension mg mL which is then encapsulated within hydrogel microspheres with diameters as small as m The hydrogel microspheres ... More
Monoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders, but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges. Hydrogel microsphere formulations offer a route to quicker, patient-friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force. Crystals of an intact monoclonal antibody are prepared as a concentrated suspension (>300 mg mL−1) which is then encapsulated within hydrogel microspheres with diameters as small as 30 µm. The hydrogel microspheres contain up to 56 wt% (dry basis) monoclonal antibody and release within 4 days under in vitro dissolution conditions. The hydrogel microspheres are concentrated into densely packed suspensions containing up to 300 mg mL−1 monoclonal antibody to evaluate their flow. These hydrogel formulations shear-thin and have lower viscosity when compared to both liquid and suspended crystal forms of the monoclonal antibody, demonstrating the potential of hydrogel microsphere encapsulants as a carrier which can mask undesirable flow properties of concentrated antibody therapeutics. Less
White spot syndrome virus WSSV the causative agent of white spot disease WSD severely affecting crustacean life forms is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target leading to various anti-cancer drugs The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase wTS Here we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate dUMP and methotrexate at a resolution of and ... More
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- and other SARS-like-CoVs encode tandem macrodomains within non-structural protein nsp The first macrodomain Mac is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose MAR from target proteins Mac likely counters host-mediated anti-viral ADP-ribosylation a posttranslational modification that is part of the host response to viral infections Mac is essential for pathogenesis in multiple animal models of CoV infection implicating it as a virulence factor and potential therapeutic target Here we report the crystal structure of SARS-CoV- Mac in complex with ADP-ribose SARS-CoV- SARS-CoV and MERS-CoV Mac exhibit similar structural folds and all proteins ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. Less
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork In eukaryotes proliferating cell nuclear antigen PCNA plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability PCNA from several organisms including Saccharomyces cerevisiae has been structurally characterised However the structural analyses of PCNA from fungal pathogens are limited Recently we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S cerevisiae Still it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic ... More
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. Less
Neurotensin receptor NTSR and related G protein coupled receptors of the ghrelin family are clinically unexploited and several mechanistic aspects of their activation and inactivation have remained unclear Enabled by a new crystallization design we present five new structures apo-state NTSR as well as complexes with nonpeptide inverse agonists SR and SR A partial agonist RTI- a and the novel full agonist SRI- providing structural rationales on how ligands modulate NTSR The inverse agonists favor a large extracellular opening of helices VI and VII undescribed so far for NTSR causing a constriction of the intracellular portion In contrast the full ... More
Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
In this thesis the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin NmHR is presented Members of the rhodopsin family such as NmHR are integral membrane proteins comprising seven helices and a retinal chromophore which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive Through photoactivation the retinal chromophore undergoes an isomerization reaction which then drives conformational changes in the protein Through variations in residue composition rhodopsin can catalyze diverse chemical reactions including pumping protons sodium or chloride ions Chloride transport is a fundamental process in biology and crucial ... More
In this thesis, the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin (NmHR) is presented. Members of the rhodopsin family, such as NmHR, are integral membrane proteins comprising seven α helices and a retinal chromophore, which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive. Through photoactivation, the retinal chromophore undergoes an isomerization reaction, which then drives conformational changes in the protein. Through variations in residue composition, rhodopsin can catalyze diverse chemical reactions, including pumping protons, sodium, or chloride ions. Chloride transport is a fundamental process in biology and crucial for maintaining the electrochemical balance of the cell.
The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.
Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.
This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less
The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.
Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.
This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less
Electron crystallography of sub-micrometre-sized D protein crystals has emerged recently as a valuable field of structural biology In meso crystallization methods utilizing lipidic mesophases particularly lipidic cubic phases LCPs can produce high-quality D crystals of membrane proteins MPs A major step towards realizing D electron crystallography of MP crystals grown in meso is to demonstrate electron diffraction from such crystals The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals Additionally the crystals have to be thin enough to let electrons traverse them without significant multiple scattering In the present ... More
Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media. Less
Structural studies of challenging targets such as G protein-coupled receptors GPCRs have accelerated during the last several years due to the development of new approaches including small-wedge and serial crystallography Here we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase LCP grown microcrystals of two human GPCRs Cysteinyl leukotriene receptors and CysLT R and CysLT R in complex with various antagonists Five datasets were collected using small-wedge synchrotron crystallography SWSX at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions Two datasets were collected using X-ray free electron laser XFEL serial ... More
Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing. Less
In this paper we applied EfficientNet a scalable deep convolution neural network with a custom data augmentation stage to a public protein crystallization image dataset called MARCO The MARCO dataset has protein crystallization images collected from several well-known institutions In our experiments EfficientNet outperformed the accuracies reported in the previous studies and it reached an overall testing and validation accuracy on the dataset Also EfficientNet achieved crystal detection accuracy in testing data which is significant improvement over existing studies
Excitatory amino acid transporters EAATs harness Na K and H gradients for fast and efficient glutamate removal from the synaptic cleft Since each glutamate is cotransported with three Na ions Na gradients are the predominant driving force for glutamate uptake We combined all-atom molecular dynamics simulations fluorescence spectroscopy and x-ray crystallography to study Na substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type Na -only bound GltPh at - resolution revealed the fully open outward-facing state primed for subsequent substrate binding Simulations and kinetic experiments established that only the binding of two Na ... More
Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations. Less
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly Vertebrate transferrins coordinate iron through interactions with two tyrosines an aspartate a histidine and a carbonate anion and conformational changes that occur upon iron binding and release have been described Much less is known about the structure and functions of insect transferrin- Tsf which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf differs from that of the vertebrate transferrins Here we report the first crystal structure resolution of an ... More
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin-1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl-lobe adopts a novel orientation and contacts with the amino-lobe. The structure revealed coordination of a single Fe3+ ion in the amino-lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans. Less
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families Here we present a functional and structural characterization of two proteins of viral rhodopsin group OLPVR and VirChR Functional analysis of VirChR shows that it is a highly selective Na K -conducting channel and in contrast to known cation channelrhodopsins it is impermeable to Ca ions We show that upon illumination VirChR is ... More
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability. Less
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase LCP However almost all previous crystallization trials have used monoacylglycerols with - cis- -octadecanoyl -rac-glycerol MO being the most widely used lipid We now report that EROCOC mixed with w w cholesterol Fig serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A A receptor A AR The structures of EROCOC -matrix grown A AR crystals were determined at resolution by serial synchrotron rotation crystallography at a cryogenic temperature and at ... More
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method. Less
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan As Lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a ... More
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan As lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes cross-linked to the matrix How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may ... More
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide–peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2,3,4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein and substrate engineering based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F A MD simulation further ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein engineering and substrate modifications based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F A MD simulation ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
- - protein isoforms regulate multiple processes in eukaryotes including apoptosis and cell division - - proteins preferentially recognize phosphorylated unstructured motifs justifying the protein-peptide binding approach to study - - phosphotarget complexes Tethering of human - - with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides simultaneously facilitating purification and crystallization Nevertheless the broader applicability to other - - isoforms and phosphopeptides was unclear Here we designed a novel - - chimera with a conserved phosphopeptide from BAD whose complex with - - is a gatekeeper of apoptosis regulation The ... More
14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the “chimeric” approach for studies of other relevant 14-3-3 complexes. Less
SARS-CoV- is a betacoronavirus virus responsible for the COVID- pandemic Here we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody CV isolated from a patient infected with SARS-CoV- in complex with the receptor binding domain RBD The structure reveals CV s epitope overlaps with the human ACE receptor binding site thus providing the structural basis for its neutralization by preventing ACE binding
There is an increasing demand for rapid effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources Here we demonstrate a compact multimodal multiphoton microscope driven by a fiber-based ultrafast laser enabling excitation wavelengths at nm and nm for nonlinear optical imaging which simultaneously records second-harmonic generation third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography which we hope ... More
There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future. Less
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia CPVT In vivo calsequestrin multimerizes into filaments but a compelling atomic-resolution structure of a calsequestrin filament is lacking We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay We also report and characterize a novel disease-associated calsequestrin mutation S I which localizes to the filament-forming interface In addition we show that a previously reported dominant disease mutation K R maps to the same multimerization surface Both mutations disrupt filamentation suggesting that dominant disease ... More
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay. We also report and characterize a novel disease-associated calsequestrin mutation, S173I, which localizes to the filament-forming interface. In addition, we show that a previously reported dominant disease mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation, suggesting that dominant disease arises from defects in multimer formation. A ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our understanding of calsequestrin biochemistry and provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias. Less
Fungal aryl-alcohol oxidases AAOx are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids with concomitant production of hydrogen peroxide H O as by-product The H O can be provided to lignin degradation pathway a biotechnological property explored in biofuel production In the thermophilic fungus Thermothelomyces thermophilus formerly Myceliophthora thermophila just one AAOx was identified in the exo-proteome
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates one from the main group M and the other from the minor group O of HIV- we identify in the C-terminal domain CTD of integrase a new functional motif constituted by four non-contiguous amino acids N K N K Mutating the lysines abolishes integration through decreased -processing and inefficient nuclear import of reverse transcribed genomes Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration The number of charges in the motif appears ... More
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3’-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. Less
Immuno-oncology approaches that utilize T cell receptors TCRs are becoming highly attractive because of their potential to target virtually all cellular proteins including cancer-specific epitopes via the recognition of peptide-human leukocyte antigen pHLA complexes presented at the cell surface However because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities efforts have been made to enhance their binding strength in some cases by several million-fold In this study we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors Additionally we performed molecular dynamics simulations ... More
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment. Less
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases EHs reveals its resourceful microscopic machinery The whole-genome sequence of Streptomyces sp CBMAI an endophytic actinobacterium isolated from Citrus sinensis branches was explored by genome mining and a putative -epoxide hydrolase named B EPH and encoded by amino acids was selected for functional and structural studies The crystal structure of B EPH was obtained at a resolution of and ... More
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/β-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Å and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications. Less
Cation-chloride cotransporters CCCs regulate the movement of chloride across membranes controlling physiological processes from cell volume maintenance to neuronal signaling Human CCCs are clinical targets for existing diuretics and potentially additional indications Here we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter Caenorhabditis elegans KCC- We observe a core a fold conserved among CCCs Using structure-based sequence alignment we analyze similarities and differences to the C-terminal domains of other CCC family members We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved suggesting ... More
Cation-chloride cotransporters (CCCs) regulate the movement of chloride across membranes, controlling physiological processes from cell volume maintenance to neuronal signaling. Human CCCs are clinical targets for existing diuretics and potentially additional indications. Here, we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter, Caenorhabditis elegans KCC-1. We observe a core a/� fold conserved among CCCs. Using structure-based sequence alignment, we analyze similarities and differences to the C-terminal domains of other CCC family members. We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved, suggesting that oligomerization and its effects may vary within the larger family. This snapshot of a eukaryotic KCC is a valuable starting point for the rational design of studies of cellular chloride regulation. Less
Pathogenic coronaviruses are a major threat to global public health as exemplified by severe acute respiratory syndrome coronavirus SARS-CoV Middle East respiratory syndrome coronavirus MERS-CoV and the newly emerged SARS-CoV- the causative agent of coronavirus disease COVID- We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus C-like protease CLpro an enzyme essential for viral replication The optimized compounds were effective against several human coronaviruses including MERS-CoV SARS-CoV and SARS-CoV- in an enzyme assay and in cell-based assays using Huh- and Vero E cell lines Two selected compounds showed antiviral effects against SARS-CoV- in cultured primary ... More
Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. Less
Ghrelin is a gastric peptide hormone with important physiological functions The unique feature of ghrelin is its Serine acyl-modification which is essential for ghrelin s activity However it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity To address these questions we solved the crystal structure of the ghrelin receptor bound to antagonist The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E and R A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap crevasse between the TM and TM bundles that is rich in ... More
Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin’s activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation. Less
T cell-mediated immunity is governed primarily by T cell receptor TCR recognition of peptide-human leukocyte antigen pHLA complexes and is essential for immunosurveillance and disease control This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region CDR loops and whereas peptide selectivity is guided by direct interactions with the TCR CDR loops Here we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A MAGE-A The TCR bound pHLA in a position shifted toward the peptide's N terminus This enabled the ... More
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. Less
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins However to date there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries To resolve this limitation we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications ... More
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies. Less
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational switching that underpin their gene-regulating capabilities Previous structural studies on the flavin mononucleotide FMN -binding aptamer of the FMN riboswitch however have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior We have determined a - resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures particularly in the conformation and orientation of the P and P helices The nearly symmetrical tertiary structure provides a mechanism ... More
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational “switching” that underpin their gene-regulating capabilities. Previous structural studies on the flavin mononucleotide (FMN)-binding aptamer of the FMN riboswitch, however, have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior. We have determined a 2.7-Å resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures, particularly in the conformation and orientation of the P1 and P4 helices. The nearly symmetrical tertiary structure provides a mechanism by which one of two pairs of adjacent helices (P3/P4 or P1/P6) undergo collinear stacking in a mutually exclusive manner, in the absence or presence of ligand, respectively. Comparison of these structures suggests the stem-loop that includes P4 and L4 is important for maintaining a global conformational state that, in the absence of ligand, disfavors formation of the P1 regulatory helix. Together, these results provide further insight to the structural basis for conformational switching of the FMN riboswitch. Less
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi Trypanosoma brucei and Leishmania major Tritryp published in opened up new perspectives for drug development targeting Chagas disease African sleeping sickness and Leishmaniasis neglected diseases affecting millions of most economically disadvantaged people Still half of the Tritryp genes code for proteins of unknown function Moreover almost of conserved eukaryotic protein domains are missing in the Tritryp genomes This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes Furthermore proteins without homologous counterparts in humans may provide ... More
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ββαββαββ topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction. Less
The vast majority of biomolecular structural information is derived from macromolecular X-ray crystallography methods which serve as a foundation for structural biology and account for nearly of the more than biomolecular structures available in the PDB Crystallography requires high-quality well-diffracting crystals coaxing biomolecules into crystalline form is a rate-limiting step in structure determination Searching for conditions in which a biomolecule will crystallize often entails screening multiple different constructs against thousands of crystallization conditions requiring large sample amounts and many person-hours in a typical laboratory set-up In recent circumstances due to the COVID- pandemic being physically in the laboratory for setting ... More
The vast majority of biomolecular structural information is derived from macromolecular X-ray crystallography
methods, which serve as a foundation for structural biology and account for nearly 90% of the more than 165,000
biomolecular structures available in the PDB. Crystallography requires high-quality, well-diffracting crystals;
coaxing biomolecules into crystalline form is a rate-limiting step in structure determination. Searching for
conditions in which a biomolecule will crystallize often entails screening multiple different constructs against
thousands of crystallization conditions, requiring large sample amounts and many person-hours in a typical
laboratory set-up. In recent circumstances due to the COVID-19 pandemic, being physically in the laboratory for
setting up crystallization screening has become even more difficult. The Crystallization Center at HWI has been in
continuous operation as a crystallization resource for 20 years providing mail-in crystallization and remote access
to crystal growth monitoring. These services have become even more critical in the face of restrictions due to
COVID-19. The Crystallization Center is a high-throughput facility that provides expertise and access to state-ofthe-
art instrumentation to facilitate efficient and cost-effective crystallization. We have extensive robotics for
automated sample handling with very small sample volumes integrated with advanced imaging and a Formulatrix
Rock Imager with SONICC for rapid detection of crystal growth. The current pipeline in the Crystallization Center
screens for 1,536 conditions in one experimental plate and employs a robust imaging schedule, all of which is then
accessible remotely. Here, we will present details about the current capacity for high-throughput crystal growth
screening. We will also discuss innovations we are developing and opportunities for enhanced crystallization
services that will further facilitate crystallization for biomolecular structure determination, including scale up and
optimization, in situ diffraction experiments and enhanced imaging for crystal detection. Less
methods, which serve as a foundation for structural biology and account for nearly 90% of the more than 165,000
biomolecular structures available in the PDB. Crystallography requires high-quality, well-diffracting crystals;
coaxing biomolecules into crystalline form is a rate-limiting step in structure determination. Searching for
conditions in which a biomolecule will crystallize often entails screening multiple different constructs against
thousands of crystallization conditions, requiring large sample amounts and many person-hours in a typical
laboratory set-up. In recent circumstances due to the COVID-19 pandemic, being physically in the laboratory for
setting up crystallization screening has become even more difficult. The Crystallization Center at HWI has been in
continuous operation as a crystallization resource for 20 years providing mail-in crystallization and remote access
to crystal growth monitoring. These services have become even more critical in the face of restrictions due to
COVID-19. The Crystallization Center is a high-throughput facility that provides expertise and access to state-ofthe-
art instrumentation to facilitate efficient and cost-effective crystallization. We have extensive robotics for
automated sample handling with very small sample volumes integrated with advanced imaging and a Formulatrix
Rock Imager with SONICC for rapid detection of crystal growth. The current pipeline in the Crystallization Center
screens for 1,536 conditions in one experimental plate and employs a robust imaging schedule, all of which is then
accessible remotely. Here, we will present details about the current capacity for high-throughput crystal growth
screening. We will also discuss innovations we are developing and opportunities for enhanced crystallization
services that will further facilitate crystallization for biomolecular structure determination, including scale up and
optimization, in situ diffraction experiments and enhanced imaging for crystal detection. Less
NKp is one of the main human natural killer NK cell activating receptors used in directed immunotherapy The oligomerization of the NKp ligand binding domain depends on the length of the C-terminal stalk region but our structural knowledge of NKp oligomerization and its role in signal transduction remains limited Moreover ligand binding of NKp is affected by the presence and type of N-glycosylation In this study we assessed whether NKp oligomerization depends on its N-glycosylation Our results show that NKp forms oligomers when expressed in HEK S GnTI cell lines with simple N-glycans However NKp was detected only as monomers ... More
NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics. Less
Sirtuins are NAD -dependent lysine deacylases which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction The human isoforms Sirt - are linked to diverse age-related diseases such as Alzheimer s Parkinson s but also cancer and AIDS Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors which often lack isoform-specificity potency or bioavailability the physiological modulation of sirtuin activity by other proteins is incompletely understood This study covers important and new aspects for the physiological ... More
Sirtuins are NAD+-dependent lysine deacylases, which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction. The human isoforms Sirt1-7 are linked to diverse age-related diseases such as Alzheimer’s, Parkinson’s, but also cancer and AIDS. Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important. While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors, which often lack isoform-specificity, potency, or bioavailability, the physiological modulation of sirtuin activity by other proteins is incompletely understood. This study covers important and new aspects for the physiological and pharmacological regulation of human Sirtuin 1. Concerning regulation by small molecules, the activation of hSirt1 by dehydroabietic acid was characterized providing potential for pharmacological modulation with a new nature-derived activator scaffold. In addition, the proposed anti-tumor potential of tranilast was linked to inhibition of hSirt1. Regarding the physiological modulation of hSirt1 activity by other proteins, conflictive results were available for hAROS. Within this thesis, hAROS was validated as hSirt1 inhibitor. Furthermore, a stable core was suggested for the intrinsically disordered protein hAROS, which will be used for future interaction analyses. Conversely, an hSirt1-activity modulating effect for human Hic1 has not been described yet. Focusing on the interaction between hSirt1 and the BTB/POZ domain of Hic1, this study showed that the interaction is limited to the catalytic domain of hSirt1 and does not require phosphorylation of hSirt1, nor the presence of NAD+ or sirtuin substrate. Hic1-BTB might even have a dual and concentration-dependent effect on hSirt1 activity, which will be subject to future studies. In addition, the first crystal structure of Hic1 was solved demonstrating a typical BTB fold and offering an analysis of putative interacting regions with hSirt1. Finally, the region of hSirt1 interacting with HIV1-Tat was also identified as the catalytic domain and an additional in vitro inhibition of the structurally similar catalytic domains of hSirt2 and hSirt3 was observed. Tat binding to hSirt1-3 was competitive to the sirtuin substrate, but not to NAD+. In line with this observation, several complex structures of hSirt3 with Tat peptides could demonstrate that acetylated or deacetylated Tat binds to the sirtuin substrate binding cleft with its basic region. Using structural superpositions and crosslinking, the major contribution to binding was found to be mediated by HIV1-Tat amino acids 49-52, while Tat amino acids 53-59 provide the additional high potency needed for physiological inhibition of hSirt1 through disruption of an important salt bridge between the hSirt1 SBD and catalytic core. Less
G-protein-coupled receptors GPCRs the largest family of cell-surface membrane proteins mediate the intracellular signal transduction of many external ligands Thus GPCRs have become important drug targets X-ray crystal structures of GPCRs are very useful for structure-based drug design SBDD Herein we produced a new antibody SRP targeting the thermostabilised apocytochrome b from Escherichia coli M W H I R L BRIL We found that a fragment of this antibody SRP Fab facilitated the crystallisation of the BRIL-tagged ligand bound GPCRs HT B and AT R Furthermore the electron densities of the ligands were resolved suggesting that SPR Fab is versatile ... More
G-protein-coupled receptors (GPCRs)�the largest family of cell-surface membrane proteins�mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them. Less
T cell recognition of peptides presented by human leukocyte antigens HLAs is mediated by the highly variable T cell receptor TCR Despite this built-in TCR variability individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD T cells Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA However few examples exist for CD T cells Here we investigate CD T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity We identify five internal epitopes that are ... More
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies. Less
Emergent nanoscale order in materials such as self-assembled lipid phases colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering SAXS Frequently residual disorder in these materials prevents high-resolution D structural characterization Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local D structure via a rich real-space distribution of three- and four-body statistics We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid revealing non-uniform curvature in the lipid channels likely due to phase separation of the lipids in the membrane Our many-body technique has general ... More
Emergent nanoscale order in materials such as self-assembled lipid phases, colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering (SAXS). Frequently, residual disorder in these materials prevents high-resolution 3D structural characterization. Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local 3D structure via a rich, real-space distribution of three- and four-body statistics. We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid, revealing non-uniform curvature in the lipid channels, likely due to phase separation of the lipids in the membrane. Our many-body technique has general applicability to nanomaterials with order in the range 10 nm−1 μm currently targeted by synchrotron SAXS and has the potential to impact diverse research areas within chemistry, biology and materials science. Less
Chemokines and their receptors mediate cell migration which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor chemokine recognition less is known about endogenous chemokine-induced receptor activation and G-protein coupling Here we present the cryo-electron microscopy structures of interleukin- IL- also known as CXCL -activated human CXC chemokine receptor CXCR in complex with Gi protein along with a crystal structure of CXCR bound to a designed allosteric antagonist Our results reveal a unique shallow mode of binding between CXCL and ... More
Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor–chemokine recognition2,3,4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles. Less
Ketol-acid reductoisomerase KARI the second enzyme in the branched-chain amino acid biosynthesis pathway is a potential drug target for bacterial infections including Mycobacterium tuberculosis Here we have screened the Medicines for Malaria Venture Pathogen Box against purified M tuberculosis Mt KARI and identified two compounds that have Ki values below nm In Mt cell susceptibility assays one of these compounds exhibited an IC value of m Co-crystallization of this compound - methylsulfonyl methyl - H-benzo b oxazin- -one MMV in complex with Staphylococcus aureus KARI which has identity with Mt KARI NADPH and Mg yielded a structure to resolution However ... More
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data. Less
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses Viral replication requires a C-like cysteine protease CLpro which processes the kDa viral polyprotein into six functional proteins The CLpro has attracted much interest due to its potential as a target for antiviral drugs A system for growing high-quality crystals of native Southampton norovirus CLpro SV CP has been established allowing the ligand-free crystal structure to be determined to in a tetrameric state This also allowed crystal-based fragment screening to be performed with various compound libraries ultimately to guide drug discovery for SV CP A total of fragments were ... More
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer. Less
The enzyme pyridoxal kinase PdxK catalyzes the conversion of pyridoxal to pyridoxal- -phosphate PLP using ATP as the co-factor The product pyridoxal- -phosphate plays a key role in several biological processes such as transamination decarboxylation and deamination In the present study full-length ORF of PdxK from Leishmania donovani LdPdxK was cloned and then purified using affinity chromatography LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH Biochemical analysis of LdPdxK with pyridoxal pyridoxamine pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK ... More
The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5′-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5′-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors. Less
Of the eighteen hemagglutinin HA subtypes H H that have been identified in bats and aquatic birds many HA subtypes have been structurally characterized However several subtypes H H and H still require characterization To better understand all of these HA subtypes at the molecular level HA structures from an A H N A swine Missouri A an A H N A turkey Ontario an A H N A duck Memphis an A H N A mallard Gurjev and an A H N A wedge-tailed shearwater Western Australia were determined by X-ray crystallography at and resolution respectively The interactions between ... More
Of the eighteen hemagglutinin (HA) subtypes (H1–H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed. Less
NS is a kDa major nonstructural protein of avian reoviruses which cause significant economic losses in the poultry industry They replicate inside viral factories in host cells and the NS protein has been suggested to be the minimal viral factor required for factory formation Thus determining the structure of NS is of great importance for understanding its role in viral infection In the study presented here a fragment consisting of residues - of NS was expressed as an EGFP fusion protein in Sf insect cells EGFP- NS - crystallization in Sf cells was monitored and verified by several imaging techniques ... More
μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the �NS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of �NS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of �NS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-�NS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-�NS(448-605) baculovirus formed rod-shaped microcrystals (5-15 �m in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 � resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 �. The final data set was merged and refined to 7.0 � resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of �NS(448-605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the �NS protein. Less
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing Co-crystallization with proteins is a straight forward approach to overcome these challenges The human RNA-binding protein U A has previously been established as crystallization module however the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U A system Here we report three crystal structures of tryptophan-containing U A variants which expand the crystallization toolbox for nucleic acids Analysis of the structures complemented by SAXS NMR spectroscopy and optical spectroscopy allow for insights into the potential of the U ... More
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes. Less
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea They possess highly diverse biological functions Explosion of research on microbial rhodopsins led to breakthroughs in their applications in particular in neuroscience An unexpected new discovery was a Na -pumping KR rhodopsin from Krokinobacter eikastus the first light-driven non-proton cation pump A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and therefore Na pumping cannot be understood in the framework ... More
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics. Less
Membrane-embedded sensor histidine kinases HKs and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment and are often crucial for their survival and pathogenicity The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms and regulate the activity of the cognate response regulator proteins with which they form two-component signaling systems TCSs Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed dubbed swinging piston helical rotation and diagonal scissoring Yet despite decades of studies there is no consensus on whether ... More
Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well. Less
Tumor-associated peptide human leukocyte antigen complexes pHLAs represent the largest pool of cell surface expressed cancer-specific epitopes making them attractive targets for cancer therapies Soluble bispecific molecules that incorporate an anti-CD effector function are being developed to redirect T cells against these targets using different approaches The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs e g immune-mobilizing monoclonal T cell receptors against cancer ImmTAC molecules whereas the second harnesses an antibody-based format TCR-mimic antibodies For both classes of reagent target specificity is vital considering the vast universe of potential pHLA molecules that can be presented on healthy ... More
Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents. Less
Ruthenium-catalysed azide alkyne cycloaddition RuAAC provides access to -disubstituted -triazole motifs in peptide engineering applications However investigation of this motif as a disulfide mimetic in cyclic peptides has been limited and the structural consequences remain to be studied We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor- NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations RMSD of the triazole linkages compared to the parent disulfide molecules The triazole-bridged peptides also displayed ... More
Ruthenium-catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics. Less