1194 Citations
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications Recently the first sodium-pumping rhodopsin KR from Krokinobacter eikastus was discovered and characterized However the existing structures of KR are contradictory and the mechanism of Na pumping is not yet understood Here we present a structure of the cationic non H light-driven pump at physiological pH in its pentameric form We also present atomic structures and functional data on the KR and its mutants including potassium pumps which show that oligomerization of the microbial ... More
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics. Less
Phox homology PX domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides markers of organelle identity in the endocytic system Although many PX domains bind the canonical endosome-enriched lipid PtdIns P others interact with alternative phosphoinositides and a precise understanding of how these specificities arise has remained elusive Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays biolayer interferometry and isothermal titration calorimetry These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns P non-specifically to various di- and tri-phosphorylated phosphoinositides bind both PtdIns ... More
Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family. Less
The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN I possesses at least putative genes encoding enzymes that belong to the -hydrolase superfamily One of those genes the metallo-hydrolase-encoding igni was cloned and heterologously expressed in Pichia pastoris The enzyme produced was purified in its catalytically active form The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of The crystal belonged to space group R with unit-cell parameters a b c It is suggested that it contains one monomer of Igni within the asymmetric unit
Inosine- -monophosphate dehydrogenase IMPDH is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents Our recent work has revealed the crucial role of one of the two structural domains i e Bateman domain in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs Thus we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH These inhibitors were shown to counteract the activation by the natural positive effector MgATP and to block ... More
Inosine-5‘-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now. Less
Poor solubility is a common challenge encountered during the development of high concentration monoclonal antibody mAb formulations but there are currently no methods that can provide predictive information on high-concentration behavior of mAbs in early discovery We explored the utility of methodologies used for determining extrapolated solubility as a way to rank-order mAbs based on their relative solubility properties We devised two approaches to accomplish this vapor diffusion technique utilized in traditional protein crystallization practice and polyethylene glycol PEG -induced precipitation and quantitation by turbidity Using a variety of in-house mAbs with known high-concentration behavior we demonstrated that both approaches ... More
Poor solubility is a common challenge encountered during the development of high concentration monoclonal antibody (mAb) formulations, but there are currently no methods that can provide predictive information on high-concentration behavior of mAbs in early discovery. We explored the utility of methodologies used for determining extrapolated solubility as a way to rank-order mAbs based on their relative solubility properties. We devised two approaches to accomplish this: 1) vapor diffusion technique utilized in traditional protein crystallization practice, and 2) polyethylene glycol (PEG)-induced precipitation and quantitation by turbidity. Using a variety of in-house mAbs with known high-concentration behavior, we demonstrated that both approaches exhibited reliable predictability of the relative solubility properties of these mAbs. Optimizing the latter approach, we developed a format that is capable of screening a large panel of mAbs in multiple pH and buffer conditions. This simple, material-saving, high-throughput approach enables the selection of superior molecules and optimal formulation conditions much earlier in the antibody discovery process, prior to time-consuming and material intensive high-concentration studies. Less
HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles This enzyme provides access to chiral -substituted alcohols that serve as building blocks in the pharmaceutical industry Wild-type HheG suffers from low thermostability which poses a significant drawback for potential applications In an attempt to thermostabilize HheG by protein engineering several single mutants at position were identified which displayed up to C increased apparent melting temperatures and up to three-fold higher activity Aromatic amino acids at position resulted even in a slightly higher enantioselectivity Crystal structures ... More
HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G. Less
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates While the impact of localization and recruitment on kinase substrate targeting can be readily assessed evaluating the relative importance of direct phosphorylation site interactions remains challenging In this study we examine the STE family of protein serine threonine kinases to investigate basic mechanisms of substrate targeting We used peptide arrays to define the phosphorylation site specificity for the majority of STE kinases and categorized them into four distinct groups Using structure-guided mutagenesis we identified key specificity-determining residues within the kinase catalytic cleft ... More
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems. Less
NEMO is an essential component in the activation of the canonical NF- B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF- B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the high-resolution structure of the unbound IKK -binding domain of NEMO that will greatly facilitate the design of NEMO IKK inhibitors The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a ... More
NEMO is an essential component in the activation of the canonical NF-?B pathway and exerts its function by recruiting the I?B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-?B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKK�-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKK�-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKK�-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes. Less
Suppressor of copper sensitivity protein C from Proteus mirabilis PmScsC is a homotrimeric disulfide isomerase that plays a role in copper tolerance which is a key virulence trait of this uropathogen Each protomer of the enzyme has an N-terminal trimerization stem residues containing a flexible linker residues connected to a thioredoxin-fold-containing catalytic domain residues Here two PmScsC variants PmScsC N and PmScsC Linker are characterized PmScsC N is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed generating a protein with dithiol oxidase rather than disulfide isomerase activity The crystal structure of PmScsC N ... More
Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance, which is a key virulence trait of this uropathogen. Each protomer of the enzyme has an N-terminal trimerization stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, two PmScsC variants, PmScsC?N and PmScsC?Linker, are characterized. PmScsC?N is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsC?N reported here reveals, as expected, a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant, PmScsC?Linker, was designed to remove the 11-amino-acid linker, and it is shown that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsC?Linker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small-angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsC?Linker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function. Less
Upon triggering by their inducer signal transduction ATPases with numerous domains STANDs initially in monomeric resting forms multimerize into large hubs that activate target macromolecules This process requires conversion of the STAND conserved core the NOD from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize In the absence of inducer autoinhibitory interactions maintain the NOD closed In particular in resting STAND proteins with an LRR- or WD -type sensor domain the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms Here we solved the first crystal structure of a ... More
Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains. Less
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors GPCRs Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A A adenosine receptor A AAR D N and S A Both structures present an overall active-like conformation however the variants show key changes in the activation motif NPxxY Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability ... More
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A2A adenosine receptor (A2AAR), D522.50N and S913.39A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G protein signaling. Utilizing the D2.50N variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery. Less
Nramp family transporters expressed in organisms from bacteria to humans enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport We present high-resolution structures of Deinococcus radiodurans Dra Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere Strikingly while metal transport requires cycling from outward- to inward-open states efficient proton transport still occurs in outward-locked but not inward-locked DraNramp We propose a model in which metal and proton enter the transporter via the same external pathway ... More
Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Less
Many drugs target the serotonin A receptor -HT AR including second-generation antipsychotics that also target the dopamine D receptor D R These drugs often produce severe side effects due to non-selective binding to other aminergic receptors Here we report the structures of human -HT AR in complex with the second-generation antipsychotics risperidone and zotepine These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket the movements of which are important for receptor activation -HT AR is structurally similar to -HT CR but possesses a unique side-extended cavity near the ... More
Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs. Less
Purple acid phosphatases PAPs are members of the large family of metallohydrolases a group of enzymes that perform a wide range of biological functions while employing a highly conserved catalytic mechanism PAPs are found in plants animals and fungi in humans they play an important role in bone turnover and are thus of interest for developing treatments for osteoporosis The majority of metallohydrolases use a metal-bound hydroxide to initiate catalysis which leads to the formation of a proposed five-coordinate oxyphosphorane species in the transition state In this work we crystallized PAP from red kidney beans rkbPAP in the presence of ... More
Purple acid phosphatases (PAPs) are members of the large family of metallohydrolases, a group of enzymes that perform a wide range of biological functions, while employing a highly conserved catalytic mechanism. PAPs are found in plants, animals and fungi; in humans they play an important role in bone turnover and are thus of interest for developing treatments for osteoporosis. The majority of metallohydrolases use a metal-bound hydroxide to initiate catalysis, which leads to the formation of a proposed five-coordinate oxyphosphorane species in the transition state. In this work, we crystallized PAP from red kidney beans (rkbPAP) in the presence of both adenosine and vanadate. The in crystallo-formed vanadate analogue of ADP provides detailed insight into the binding mode of a PAP substrate, captured in a structure that mimics the putative fivecoordinate transition state. Our observations not only provide unprecedented insight into the mechanism of metallohydrolases, but might also guide the structure-based design of inhibitors for application in the treatment of several human illnesses. Less
Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures However a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments Here iterative screen optimization ISO a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment is described After designing a novel high-throughput crystallization screen to take full advantage of this method the value of ISO is demonstrated by using it to successfully crystallize a ... More
Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full advantage of this method, the value of ISO is demonstrated by using it to successfully crystallize a panel of six diverse proteins. The results suggest that ISO is an effective method to obtain macromolecular crystals, particularly for proteins that crystallize under a narrow range of precipitant concentrations. Less
Knowledge of protein behavior stability during freeze thaw FT operations is essential for storage and production processes in the biopharmaceutical industry FT stress involves freeze concentration cold denaturation and ice crystals formation which can result in protein aggregation Therefore it is important to understand the ongoing FT processes and the influence of different solution parameters In order to evaluate the ongoing processes during FT up to C phase diagrams with lysozyme from chicken egg white and sodium chloride were generated Thereby three different buffer systems with varying buffer substances and ionic strengths at pH and pH were investigated As indicators ... More
Knowledge of protein behavior/stability during freeze/thaw (FT) operations is essential for storage and production processes in the biopharmaceutical industry. FT stress involves freeze concentration, cold denaturation, and ice crystals formation which can result in protein aggregation. Therefore, it is important to understand the ongoing FT processes, and the influence of different solution parameters. In order to evaluate the ongoing processes during FT (up to −80°C), phase diagrams with lysozyme from chicken egg white and sodium chloride were generated. Thereby, three different buffer systems with varying buffer substances and ionic strengths at pH 3 and pH 5 were investigated. As indicators for the ongoing FT processes, the phase behavior, crystal morphology and solubility were used. An increased number of cycles led, for example, to the formation of micro crystals, sea urchin crystals – indicating LLPS and/or high supersaturation – and precipitate. Furthermore, the buffer substances had a more distinct influence on the phase behavior and morphology compared to the ionic strength differences. The solubility line itself was only shifted when distinct changes in the phase behavior could be observed. In summary, a tool was developed for using the phase behavior and especially the crystal morphology as indicator for underlying processes during FT operations. Less
Mycobacterium tuberculosis Mtb the main causative agent of tuberculosis TB is naturally resistant to -lactam antibiotics due to the production of the extended spectrum -lactamase BlaC -Lactam -lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB However still little is known of the exact mechanism of BlaC inhibition by the -lactamase inhibitors currently approved for clinical use clavulanic acid sulbactam tazobactam and avibactam Here we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors The Da adduct derived from clavulanate and the trans-enamine ... More
Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other β-lactamases are discussed. Less
VMXi is a new high-flux microfocus macromolecular crystallography beamline at Diamond Light Source The beamline dedicated to fully automated and fully remote data collection of macromolecular crystals in situ allows rapid screening of hundreds of crystallization plates from multiple user groups Its main purpose is to give fast feedback at the complex stages of crystallization and crystal optimization but it also enables data collection of small and delicate samples that are particularly difficult to harvest using conventional cryo-methods crystals grown in the lipidic cubic phase and allows for multi-crystal data collections in drug discovery programs The beamline is equipped with ... More
VMXi is a new high-flux microfocus macromolecular crystallography beamline at Diamond Light Source. The beamline, dedicated to fully automated and fully remote data collection of macromolecular crystals in situ, allows rapid screening of hundreds of crystallization plates from multiple user groups. Its main purpose is to give fast feedback at the complex stages of crystallization and crystal optimization, but it also enables data collection of small and delicate samples that are particularly difficult to harvest using conventional cryo-methods, crystals grown in the lipidic cubic phase, and allows for multi-crystal data collections in drug discovery programs. The beamline is equipped with two monochromators: one with a narrow band-pass and fine energy resolution (optimal for regular oscillation experiments), and one with a wide band-pass and a high photon flux (optimal for fast screening). The beamline has a state-of-the-art detector and custom goniometry that allows fast data collection. This paper describes the beamline design, current status and future plans. Less
Rab proteins belong to the ras superfamily of small GTPases and play important roles in the regulation of vesicular transport within the eukaryaotic cell The central mechanistic hallmark of all GTPases is their ability to bind the nucleotide GTP and to hydrolyze it to GDP Dependent on the nucleotide state small GTPases can take specific conformations which serve different roles GTP-bound small GTPases can interact with so called effector proteins and thereby actively mediate a specific function whereas in their GDP-bound state they are inactive Due to their ability to cycle between an active and inactive state small GTPases are ... More
Rab proteins belong to the ras superfamily of small GTPases and play important roles in the regulation of vesicular transport within the eukaryaotic cell. The central mechanistic hallmark of all GTPases is their ability to bind the nucleotide GTP and to hydrolyze it to GDP. Dependent on the nucleotide state small GTPases can take specific conformations which serve different roles: GTP-bound small GTPases can interact with so called effector proteins and thereby actively mediate a specific function, whereas in their GDP-bound state, they are inactive. Due to their ability to cycle between an active and inactive state, small GTPases are often called „molecular switches“. In order to control their activity in a spatially and temporally exact manner, additional proteins are necessary: guanine nucleotide exchange factors (short: GEFs) and GTPase activating proteins (short: GAPs). While GEFs facilitate the exchange of GDP for GTP and thereby activate the associated GTPase, GAPs stimulate the hydrolysis of GTP to GDP and thereby inactivate the GTPase. As for any GTPase the knowledge of the regulatory context of a Rab protein is thus crucial to fully understand how it exerts its function. However, although over 60 human Rab proteins have been identified so far, comparatively little is known about the regulation of Rab proteins by their GEFs, since only few Rab-GEFs have been identified. The main reason for this is that the identification of Rab-GEFs by in silico approaches which search for cognate genes has been hampered by the huge diversity of structures and sequences of Rab-GEFs. In order to facilitate the identification of new GEFs for Rab proteins this dissertation presents a protocol that has been adapted and optimized to perform specific pull-down experiments for GEFs. It exploits the enzymatic mechanism of GEFs by stabilizing an intermediate, nucleotide-free state of GTPases in which they have a very high affinity towards their GEF, favoring their enrichment in the pull-down experiments. Evidence of the protocol’s applicability is given within this dissertation using the known Rab/GEFcouple Sec4/Sec2 as an example. To correlate experimental observations of G-proteins with a defined nucleotide state in vitro, one can use non-hydrolyzable nucleotide analogs such as GppNHp. In vivo, however, these analogs are prone to be exchanged with intracellular nucleotides. Alternative strategies for creating constitutive active or inactive G-proteins are often of dubious efficiency or charged with artefacts. In order to gain definitive control over a G-protein’s nucleotide state, the research group of Prof. Roger Goody has developed a new kind of nucleotide analogs which can be covalently linked to the G-protein. The covalent bond prevents nucleotide exchange and ensures a defined nucleotide state. Based on x-ray crystallographic analyses it is shown within this thesis that the modification of the small GTPase Ypt7 with the GTP variant of this new kind of nucleotides does not disturb the structure of Ypt7. Less
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions Here we performed deep single-cell RNA sequencing scRNA-seq of microglia and related myeloid cells sorted from various regions of embryonic early postnatal and adult mouse brains We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes regardless of brain region By contrast early postnatal microglia are more heterogeneous We discovered a proliferative-region-associated microglia PAM ... More
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease. Less
With the advent of X-Ray free electron lasers FELs the field of serial femtosecond crystallography SFX was borne allowing a stream of nanocrystals to be measured individually and diffraction data to be collected and merged to form a complete crystallographic data set This allows submicron to micron crystals to be utilized in an experiment when they were once at best only an intermediate result towards larger usable crystals SFX and its variants have opened new possibilities in structural biology including studies with increased temporal resolution extending to systems with irreversible reactions and minimizing artifacts related to local radiation damage Perhaps ... More
With the advent of X-Ray free electron lasers (FELs), the field of serial femtosecond crystallography (SFX) was borne, allowing a stream of nanocrystals to be measured individually and diffraction data to be collected and merged to form a complete crystallographic data set. This allows submicron to micron crystals to be utilized in an experiment when they were once, at best, only an intermediate result towards larger, usable crystals. SFX and its variants have opened new possibilities in structural biology, including studies with increased temporal resolution, extending to systems with irreversible reactions, and minimizing artifacts related to local radiation damage. Perhaps the most profound aspect of this newly established field is that �molecular movies,� in which the dynamics and kinetics of biomolecules are studied as a function of time, are now an attainable commodity for a broad variety of systems, as discussed in Chaps. 11 and 12. However, one of the historic challenges in crystallography has always been crystallogenesis and this is no exception when preparing samples for serial crystallography methods. In the following chapter, we focus on some of the specific characteristics and considerations inherent in preparing a suitable sample for successful serial crystallographic approaches. Less
Binding between DIP and Dpr neuronal-recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system Each lamina neuron was previously shown to express many Dprs Here we demonstrate by contrast that their synaptic partners typically express one or two DIPs with binding specificities matched to the lamina neuron-expressed Dprs A deeper understanding of the molecular logic of DIP Dpr interaction requires quantitative studies on the properties of these proteins We thus generated a quantitative affinity-based DIP Dpr interactome for all DIP Dpr protein family members This revealed a broad range of ... More
Binding between DIP and Dpr neuronal-recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo. Less
Virtually all terrestrial habitats are dominated by angiosperms or flowering plants Their success in colonizing new habitats and supplanting other species is due to the advent of a complex reproductive structure the flower The flower unites the male and female organs into one compact structure and encloses the seed Flowering plants are not only the dominant type of land plants but also are the primary source of food and habitat for all animals including humans In evolutionary terms flowers are considered a recent development and have been a subject of speculation from the time of Charles Darwin who termed the ... More
Virtually all terrestrial habitats are dominated by angiosperms, or flowering plants. Their
success in colonizing new habitats and supplanting other species is due to the advent of a
complex reproductive structure � the flower. The flower unites the male and female organs
into one compact structure and encloses the seed. Flowering plants are not only the dominant
type of land plants, but also are the primary source of food and habitat for all animals,
including humans. In evolutionary terms, flowers are considered a recent development and
have been a subject of speculation from the time of Charles Darwin who termed the dominant
rise and diversification of flowering plants as �an abominable mystery�* due to the lack of a
smooth transition from non-flowering to flowering plants in the fossil record. With the
sequencing of multiple genomes from gymnosperms (non-flowering seed plants), basal
angiosperms and higher flowering plants, certain gene families have been identified which
play a central role in the development and evolution of the flower. My research focuses on
one such family of high-level regulators, the MADS transcription factor (TF) family. This TF
family helps to orchestrate flower development among other functions. As such, there is great
interest in understanding the molecular mechanisms of the MADS family and how these
proteins are able to control complex reproductive pathways.
This project integrates different biophysical techniques including x-ray crystallography,
small angle x-ray scattering (SAXS) and atomic force microscopy (AFM) to investigate
protein-protein and protein-DNA interactions of MADS TFs. No studies to date have
investigated the molecular mechanisms of MADS TFs using this integrated structural
approach.
One important hurdle in the study of the MADS TFs has been recombinant protein
expression and purification. In this project, recombinant purification protocols for several
full length MADS TFs were established, allowing the structural and biochemical
characterisation of the proteins. The crystal structure of the oligomerisation domain of the
MADS family protein SEPALLATA3 (SEP3) is presented and used as a template for
understanding the oligomerisation patterns of the larger family and the molecular basis for
protein-protein interactions. Investigation of solution structures, derived from SAXS studies,
of AGAMOUS (AG) and SHORT VEGETATIVE PHASE (SVP) along with biochemical
characterisation of their oligomerisation states are also presented.
In order to study protein-DNA interactions, complementary methods were used. An
important putative property of the MADS TFs is their ability to change the structure of DNA
through the formation of DNA loops. MADS TFs are hypothesized to oligomerise and bind
DNA at two different sites, potentiating looping of DNA. Using AFM, the first direct
evidence of DNA looping by SEP3 is described. The DNA binding characteristics of SVP
were studied using electrophoretic mobility shift assay (EMSA), microscale thermophoresis
(MST) and AFM. Unlike SEP3, SVP is dimeric and thus exhibits different DNA-binding
patterns.
The data presented here provide an atomic and structural basis for MADS TF function.
Based on this work, we now are beginning to understand some of the oligomerisation and
DNA-binding specificity determinants. These studies demonstrate how the MADS TFs
oligomerise and the results show that we can disrupt oligomerisation and potentially DNAbinding very specifically through the introduction of point mutations. Future work will
investigate the in vivo consequences of altered oligomerisation and how this affects different
developmental programs in plant reproduction and floral organ morphogenesis. Less
success in colonizing new habitats and supplanting other species is due to the advent of a
complex reproductive structure � the flower. The flower unites the male and female organs
into one compact structure and encloses the seed. Flowering plants are not only the dominant
type of land plants, but also are the primary source of food and habitat for all animals,
including humans. In evolutionary terms, flowers are considered a recent development and
have been a subject of speculation from the time of Charles Darwin who termed the dominant
rise and diversification of flowering plants as �an abominable mystery�* due to the lack of a
smooth transition from non-flowering to flowering plants in the fossil record. With the
sequencing of multiple genomes from gymnosperms (non-flowering seed plants), basal
angiosperms and higher flowering plants, certain gene families have been identified which
play a central role in the development and evolution of the flower. My research focuses on
one such family of high-level regulators, the MADS transcription factor (TF) family. This TF
family helps to orchestrate flower development among other functions. As such, there is great
interest in understanding the molecular mechanisms of the MADS family and how these
proteins are able to control complex reproductive pathways.
This project integrates different biophysical techniques including x-ray crystallography,
small angle x-ray scattering (SAXS) and atomic force microscopy (AFM) to investigate
protein-protein and protein-DNA interactions of MADS TFs. No studies to date have
investigated the molecular mechanisms of MADS TFs using this integrated structural
approach.
One important hurdle in the study of the MADS TFs has been recombinant protein
expression and purification. In this project, recombinant purification protocols for several
full length MADS TFs were established, allowing the structural and biochemical
characterisation of the proteins. The crystal structure of the oligomerisation domain of the
MADS family protein SEPALLATA3 (SEP3) is presented and used as a template for
understanding the oligomerisation patterns of the larger family and the molecular basis for
protein-protein interactions. Investigation of solution structures, derived from SAXS studies,
of AGAMOUS (AG) and SHORT VEGETATIVE PHASE (SVP) along with biochemical
characterisation of their oligomerisation states are also presented.
In order to study protein-DNA interactions, complementary methods were used. An
important putative property of the MADS TFs is their ability to change the structure of DNA
through the formation of DNA loops. MADS TFs are hypothesized to oligomerise and bind
DNA at two different sites, potentiating looping of DNA. Using AFM, the first direct
evidence of DNA looping by SEP3 is described. The DNA binding characteristics of SVP
were studied using electrophoretic mobility shift assay (EMSA), microscale thermophoresis
(MST) and AFM. Unlike SEP3, SVP is dimeric and thus exhibits different DNA-binding
patterns.
The data presented here provide an atomic and structural basis for MADS TF function.
Based on this work, we now are beginning to understand some of the oligomerisation and
DNA-binding specificity determinants. These studies demonstrate how the MADS TFs
oligomerise and the results show that we can disrupt oligomerisation and potentially DNAbinding very specifically through the introduction of point mutations. Future work will
investigate the in vivo consequences of altered oligomerisation and how this affects different
developmental programs in plant reproduction and floral organ morphogenesis. Less
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging due to its affordability stability ease of administration and clinical efficacy However misoprostol lacks receptor and tissue selectivities and thus its use is accompanied by a number of serious side-effects The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction Here we present the resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E receptor EP The active-state structure reveals a completely enclosed ... More
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging due to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities and thus its use is accompanied by a number of serious side-effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active-state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol ring structure. Modelling of selective agonists in EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings. Less
Prostanoids are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G-protein-coupled receptors GPCRs Here we report the crystal structure of human prostaglandin PG E receptor subtype EP bound to endogenous ligand PGE at resolution The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands
Prostaglandin E receptor EP a G-protein-coupled receptor is involved in disorders such as cancer and autoimmune disease Here we report the crystal structure of human EP in complex with its antagonist ONO-AE - and an inhibitory antibody at resolution The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer proximal to the highly conserved Arg residue in the seventh transmembrane domain Functional and docking studies demonstrate that the natural agonist PGE binds in a similar manner This structural information also provides insight into the ligand ... More
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family. Less
Toxin antitoxin TA systems are involved in diverse physiological processes in prokaryotes but their exact role in Mycobacterium tuberculosis Mtb virulence and in vivo stress adaptation has not been extensively studied Here we demonstrate that the VapBC TA module is essential for Mtb to establish infection in guinea pigs RNA-sequencing revealed that overexpression of VapC toxin results in metabolic slowdown suggesting that modulation of the growth rate is an essential strategy for in vivo survival Interestingly overexpression of VapC resulted in the upregulation of chromosomal TA genes suggesting the existence of highly coordinated crosstalk among TA systems In this study ... More
Toxin–antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin–substrate and toxin–antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb. Less
URI http hdl handle net Content Type Thesis Files Elion-Jourard Shira S MSc thesis pdf Permanent link https hdl handle net
Metallo- -Lactamases MBLs protect bacteria from almost all -lactam antibiotics Verona integron-encoded MBL VIM enzymes are among the most clinically important MBLs with VIM- increasing in carbapenem-resistant Enterobacteriaceae Escherichia coli Klebsiella pneumoniae that are among the hardest bacterial pathogens to treat VIM enzymes display sequence variation at residues and that in related MBLs are conserved and participate in substrate binding How they accommodate this variability while retaining catalytic efficiency against a broad substrate range has remained unclear Here we present crystal structures of VIM- and its complexes with a substrate-mimicking thioenolate inhibitor ML F that restores meropenem activity against a ... More
Metallo-β-Lactamases (MBLs) protect bacteria from almost all β-lactam antibiotics. Verona integron-encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM-1 increasing in carbapenem-resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM-1 and its complexes with a substrate-mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM-1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water-mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221-bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs. Less
Human respiratory syncytial virus HRSV is a negative-stranded RNA virus that causes a globally prevalent respiratory infection which can cause lifethreatening illness particularly in the young elderly and immunocompromised HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase RdRp For replication this complex comprises the phosphoprotein P and the large protein L whereas for transcription the M - protein is also required M - is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M - surface such that binding of these ... More
Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause lifethreatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90 – 110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences. Less
Human muscarinic receptor M is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors Muscarinic receptors are targets for multiple neurodegenerative diseases The challenge has been designing subtype selective ligands against one of the five muscarinic receptors We report high resolution structures of a thermostabilized mutant M receptor bound to a subtype selective antagonist AF-DX and a non-selective antagonist NMS The thermostabilizing mutation S R in M was predicted using a theoretical strategy previously developed in our group Comparison of the crystal structures and pharmacological properties of the M receptor shows that the ... More
Human muscarinic receptor, M2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptor compared to M3 receptor leads to subtype selectivity of AF-DX 384. Less
Kinesin- transports numerous cellular cargoes along microtubules The kinesin- light chain KLC mediates cargo binding and regulates kinesin- motility To investigate the molecular basis for kinesin- recruitment and activation by cargoes we solved the crystal structure of the KLC tetratricopeptide repeat TPR domain bound to the cargo JIP This combined with biophysical and molecular evolutionary analyses reveals a kinesin- cargo binding site located on KLC TPR which is conserved in homologs from sponges to humans In the complex JIP crosslinks two KLC TPR domains via their TPR s We show that TPR forms a dimer interface that mimics JIP binding ... More
Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms. Less
Chagas disease caused by Trypanosoma cruzi affects millions of people in South America and no satisfactory therapy exists especially for its life threatening chronic phase We targeted the Proline Racemase of T cruzi which is present in all stages of the parasite life cycle to discover new inhibitors against this disease The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design In previous work to break through the chemical space afforded to virtual screening and drug design we generated intermediate models between the open ligand free and closed ... More
Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific—stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design. Less
The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits whether within pockets of interest or merely on surface sites this is the key information for structure-based design and for enabling synthesis of follow-up molecules Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data in academic and industry groups generated datasets of uniquely soaked ... More
The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program. The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites: this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data: in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals. It has also generated a large number of learnings concerning the main remaining bottleneck, namely, obtaining a suitable crystal system that will support a successful fragment screen. Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab. Less
Inteins remove themselves from a precursor protein by protein splicing Due to the concomitant structural changes of the host protein this self-processing reaction has enabled many applications in protein biotechnology and chemical biology We show that the evolved M mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking extein position compared to the parent intein in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein Surprisingly side chains with increased steric bulk compared to the native Gly residue including D-amino acids were found to compensate for ... More
Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins. Less
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than cells from organs and tissues These data represent a new resource for cell biology reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues such as T lymphocytes and endothelial cells from different anatomical locations Two distinct technical approaches were used for most organs one approach microfluidic droplet-based -end counting enabled the survey of thousands of cells at relatively low coverage whereas the other full-length ... More
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3′-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology. Less
A statistical model enables auto-calibration of second harmonic generation SHG images for quantifying trace crystallinity within amorphous solid dispersions ASDs over a wide dynamic range of crystallinity In this paper we demonstrate particle-counting approaches for quantifying trace crystallinity combined with analytical expressions correcting for particle overlap bias in higher crystallinity regimes to extend the continuous dynamic range of standard particle-counting algorithms through to the signal averaging regime The reliability of the values recovered by these expressions was demonstrated with simulated data as well as experimental data obtained for an amorphous solid dispersion formulation containing evacetrapib an Eli Lilly and Company ... More
A statistical model enables auto-calibration of second harmonic generation (SHG) images for quantifying trace crystallinity within amorphous solid dispersions (ASDs) over a wide dynamic range of crystallinity. In this paper, we demonstrate particle-counting approaches for quantifying trace crystallinity, combined with analytical expressions correcting for particle overlap bias in higher crystallinity regimes to extend the continuous dynamic range of standard particle-counting algorithms through to the signal averaging regime. The reliability of the values recovered by these expressions was demonstrated with simulated data as well as experimental data obtained for an amorphous solid dispersion formulation containing evacetrapib, an Eli Lilly and Company compound. Since particle counting independently recovers the crystalline volume and the SHG intensity, the average SHG intensity per unit volume can be used as an internal calibrant for quantifying crystallinity at higher volume fractions, for which particle counting is no longer applicable. Less
The closely related type III secretion system zinc metalloprotease effector proteins GtgA GogA and PipA are translocated into host cells during Salmonella infection They then cleave nuclear factor -light-chain-enhancer of activated B cells NF- B transcription factor subunits dampening activation of the NF- B signaling pathway and thereby suppressing host immune responses We demonstrate here that GtgA GogA and PipA cleave a subset of NF- B subunits including p RelB and cRel but not NF- B and NF- B whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF- B ... More
The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1′ residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1′ residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence. Less
Purine nucleoside phosphorylases PNPs play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway Here we present the structural and kinetic characterization of a new PNP isoform from S mansoni named as SmPNP Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands The binding of cytosine was confirmed by isothermal titration calorimetry with a KD of M and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of M SmPNP also displays catalytic activity against inosine and adenosine making it ... More
Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 μM, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 μM. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity. Less
Neisserial heparin binding antigen NHBA is one of three main recombinant protein antigens in CMenB a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region an arginine-rich region that binds heparin and a C-terminal domain that folds as an anti-parallel -barrel and that upon release after cleavage by human proteases alters endothelial permeability NHBA induces bactericidal antibodies in humans and NHBA-specific antibodies elicited by the CMenB vaccine contribute to serum bactericidal activity the correlate of protection To better understand the structural bases of ... More
Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel β-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal β-hairpin fragment and the β-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the β-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects. Less
Frizzled receptors FZDs are class-F G-protein-coupled receptors GPCRs that function in Wnt signalling and are essential for developing and adult organisms As central mediators in this complex signalling pathway FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research Here we present an atomic-resolution structure of the human Frizzled receptor FZD transmembrane domain in the absence of a bound ligand The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed and is distinct from all other GPCR structures reported so far Within this ... More
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs. Less
The avian influenza A H N virus continues to cause human infections in China and is a major ongoing public health concern Five epidemic waves of A H N infection have occurred since and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves Moreover highly pathogenic avian influenza HPAI A H N viruses were also isolated during the fifth epidemic wave Here we present a detailed structural and biochemical analysis of the surface hemagglutinin HA antigen ... More
The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that, compared to the 2013 virus HAs, the fifth-wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9) HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth-wave H7 HAs analyzed in this study. Less
Whirlin is a protein essential to sensory neurons Its defects are responsible for nonsyndromic deafness or for the Usher syndrome a condition associating congenital deafness and progressive blindness This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells The HHD domain of whirlin is the only domain shared by all isoforms but its function remains unknown In this article we report its crystal structure in two distinct conformations a monomeric five-helix bundle similar to the known structure of other HHD ... More
Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five-helix bundle, similar to the known structure of other HHD domains, and a three-helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five-helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain. Less
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers FELs for serial femtosecond crystallography measurements The high X-ray intensity in the focus initiates an explosion of the microjet and sample With the advent of X-ray FELs with megahertz rates the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using nm radiation The operation of gas-dynamic nozzles that ... More
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments. Less
Automation provides a number of benefits to clinical industrial and research laboratories including elimination of human errors improvement in consistency minimization of contamination increased throughput decreased costs and reduced hands-on time At the same time multiplex assays offer the benefits of more data per sample faster reaction times reduced sample consumption enhanced scalability and a number of additional benefits due to elimination of repetitive steps Integrating laboratory automation with multiplex assays provides significant efficiencies to workflow processes especially when a large number of samples are being analyzed or when a highly multiplexed assay is being developed Automation can be partially ... More
Automation provides a number of benefits to clinical, industrial, and research laboratories, including elimination of human errors, improvement in consistency, minimization of contamination, increased throughput, decreased costs, and reduced hands-on time. At the same time, multiplex assays offer the benefits of more data per sample, faster reaction times, reduced sample consumption, enhanced scalability, and a number of additional benefits due to elimination of repetitive steps. Integrating laboratory automation with multiplex assays provides significant efficiencies to workflow processes, especially when a large number of samples are being analyzed, or when a highly multiplexed assay is being developed. Automation can be partially or fully implemented. Less
Rotaviruses RVs cause life-threatening diarrhea in infants and children worldwide Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens HBGA as both cell attachment and susceptibility factors for the globally dominant P P and P genotypes of human RVs How these genotypes interact with HBGA is not known Here our crystal structures of P and a neonate-specific P VP s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs consistent with their prevalence Remarkably the VP ... More
Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P[6] RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut. Less
Angiotensin II AngII plays a central role in regulating human blood pressure which is mainly mediated by interactions between AngII and the G-protein-coupled receptors GPCRs AngII type receptor AT R and AngII type receptor AT R We have solved the crystal structure of human AT R binding the peptide ligand Sar Ile AngII and its specific antibody at - resolution Sar Ile AngII interacts with both the core binding domain where the small-molecule ligands of AT R and AT R bind and the extended binding domain which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor We ... More
Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the ‘core’ binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the ‘extended’ binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs. Less
The chemokine receptor CCR is an important anti-HIV human immunodeficiency virus drug target owning to its pivotal role in HIV- viral entry as a co-receptor Here we present a resolution crystal structure of CCR bound to PF- a second-generation oral CCR antagonist currently in phase II clinical trials PF- and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino N - and carboxyl C - substituents Comparison of the CCR PF- structure with the previously determined structure of CCR in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes ... More
The chemokine receptor CCR5 is an important anti-HIV (human immunodeficiency virus) drug target owning to its pivotal role in HIV-1 viral entry as a co-receptor. Here, we present a 2.9 Å resolution crystal structure of CCR5 bound to PF-232798, a second-generation oral CCR5 antagonist currently in phase II clinical trials. PF-232798 and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino (N)- and carboxyl (C)- substituents. Comparison of the CCR5–PF-232798 structure with the previously determined structure of CCR5 in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes of the receptor. Our results not only offer insights into the phenomenon that PF-232798 has higher affinity and alternative resistance profile to maraviroc, but also will facilitate the design of new anti-HIV drugs. Less
The Machine Recognition of Crystallization Outcomes MARCO initiative has assembled roughly half a million annotated images of macromolecular crystallization experiments from various sources and setups Here state-of-the-art machine learning algorithms are trained and tested on different parts of this data set We find that more than of the test images can be correctly labeled irrespective of their experimental origin Because crystal recognition is key to high-density screening and the systematic analysis of crystallization experiments this approach opens the door to both industrial and fundamental research applications
CCR is the primary chemokine receptor utilized by HIV to infect leukocytes whereas CCR ligands inhibit infection by blocking CCR engagement with HIV gp To guide the design of improved therapeutics we solved the structure of CCR in complex with chemokine antagonist P CCL Several structural features appeared to contribute to the anti-HIV potency of P CCL including the distinct chemokine orientation relative to the receptor the near-complete occupancy of the receptor binding pocket the dense network of intermolecular hydrogen bonds and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc Molecular modeling indicated that HIV gp mimicked ... More
CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases. Less