434 Citations
Balanced fusion and fission are key for the proper function and physiology of mitochondria Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance Mgm in fungi or the related protein optic atrophy OPA in animals Mgm is required for the preservation of mitochondrial DNA in yeast whereas mutations in the OPA gene in humans are a common cause of autosomal dominant optic atrophy a genetic disorder that affects the optic nerve Mgm and OPA are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space ... More
Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3,4,5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy—a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane—not the inner membrane—fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane. Less
The -oxopurine phosphoribosyltransferases PRTs are drug targets for the treatment of parasitic diseases This is due to the fact that parasites are auxotrophic for the -oxopurine bases relying on salvage enzymes for the synthesis of their -oxopurine nucleoside monophosphates In Trypanosoma brucei the parasite that is the aetiological agent for sleeping sickness there are three -oxopurine PRT isoforms Two are specific for hypoxanthine and guanine whilst the third characterized here uses all three naturally occurring bases with similar efficiency Here we have determined crystal structures for TbrHGXPRT in complex with GMP XMP and IMP to investigate the structural basis for ... More
The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. Less
The structure of BgaR a transcriptional regulator of the lactose operon in Clostridium perfringens has been solved by SAD phasing using a mercury derivative BgaR is an exquisite sensor of lactose with a binding affinity in the low-micromolar range This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form and was compared with AraC another saccharide-binding transcriptional regulator It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold but that part of the saccharide is exposed to bulk solvent This differs from the classical ... More
The structure of BgaR, a transcriptional regulator of the lactose operon in�Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC. Less
The universally conserved N -threonylcarbamoyladenosine t A modification of tRNA is essential for translational fidelity In bacteria t A biosynthesis starts with the TsaC TsaC -catalyzed synthesis of the intermediate threonylcarbamoyl adenylate TC AMP followed by transfer of the threonylcarbamoyl TC moiety to adenine- of tRNA by the TC-transfer complex comprised of TsaB TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t A cycle We report a - crystal structure of the T maritima TC-transfer complex TmTsaB D E bound to Mg -ATP in the ATPase site and substrate analog carboxy-AMP in the TC-transfer ... More
The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system. Less
The HLA-A -restricted decapeptide EAAGIGILTV derived from melanoma antigen recognized by T-cells- MART- protein represents one of the best-studied tumor associated T-cell epitopes but clinical results targeting this peptide have been disappointing This limitation may reflect the dominance of the nonapeptide AAGIGILTV at the melanoma cell surface The decapeptide and nonapeptide are presented in distinct conformations by HLA-A and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly Here we studied the MEL TCR that potently recognizes the nonapeptide The structure of the MEL -HLA-A -AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide MHC ... More
The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells. Less
The human protein tyrosine phosphatase non-receptor type PTPN is a PDZ PSD- Dlg ZO- domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers Interestingly the high-risk genital human papillomavirus HPV types and target the PDZ domain of PTPN The presence of a PDZ binding motif PBM on E confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential Here we report the molecular basis of interaction between the PDZ domain of PTPN and the PBM of the HPV E protein We combined biophysical NMR and X-ray experiments to ... More
The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a PDZ (PSD-95/Dlg/ZO-1) domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers. Interestingly, the high-risk genital human papillomavirus (HPV) types 16 and 18 target the PDZ domain of PTPN3. The presence of a PDZ binding motif (PBM) on E6 confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential. Here, we report the molecular basis of interaction between the PDZ domain of PTPN3 and the PBM of the HPV E6 protein. We combined biophysical, NMR and X-ray experiments to investigate the structural and functional properties of the PDZ domain of PTPN3. We showed that the C-terminal sequences from viral proteins encompassing a PBM interact with PTPN3-PDZ with similar affinities to the endogenous PTPN3 ligand MAP kinase p38γ. PBM binding stabilizes the PDZ domain of PTPN3. We solved the X-ray structure of the PDZ domain of PTPN3 in complex with the PBM of the HPV E6 protein. The crystal structure and the NMR chemical shift mapping of the PTPN3-PDZ/peptide complex allowed us to pinpoint the main structural determinants of recognition of the C-terminal sequence of the E6 protein and the long-range perturbations induced upon PBM binding. Less
The polymerase of negative-stranded RNA viruses consists of the large protein L and the phosphoprotein P the latter serving both as a chaperon and a cofactor for L We mapped within measles virus MeV P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain MD of P is required for gene expression MeV MD is kinked as a result of the presence of a stammer Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay By contrast P activity is rather tolerant of substitutions ... More
The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the “a” or “d” hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness. Less
The three members of the endocrine fibroblast growth factor FGF family designated FGF FGF and FGF mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor FGFR bound to either -Klotho or -Klotho receptors Structural analyses of ligandoccupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF and FGF to -Klotho or -Klotho respectively They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling Here we describe the crystal structure the C-terminal tail of ... More
The three members of the endocrine fibroblast growth factor (FGF) family designated FGF19, FGF21, and FGF23 mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor (FGFR) bound to either α-Klotho or β-Klotho receptors. Structural analyses of ligandoccupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF21 and FGF23 to β-Klotho or α-Klotho, respectively. They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling. Here we describe the crystal structure the C-terminal tail of FGF19 (FGF19CT) bound to sKLB and demonstrate that FGF19CT and FGF21CT bind to the same binding site on sKLB, via a multiturn D-P motif to site 1 and via a SP-S motif to the pseudoglycoside hydrolase region (site 2). Binding affinities to sKLB and cellular stimulatory activities of FGF19CT, FGF21CT, and a variety of chimeric mutants to cells expressing β-Klotho together with FGFR1c or FGFR4 were also analyzed. These experiments as well as detailed comparison of the structures of free and ligand-occupied sKLB to the structure of ligandoccupied sKLA reveal a general mechanism for recognition of endocrine FGFs by Klotho proteins and regulatory interactions with FGFRs that control their pleiotropic cellular responses. Less
Many long-standing image processing problems in applied science domains are finding solutions through the application of deep learning approaches to image processing Here we present one such application the case of classifying images of protein crystallisation droplets The Collaborative Crystallisation Centre in Melbourne Australia is a medium throughput service facility that produces between five and twenty thousand images per day This submission outlines a reliable and robust machine learning pipeline that autonomously classifies these images using CSIRO s high-performance computing facilities Our pipeline achieves improved accuracies over existing implementations and delivers these results in real time We discuss the specific ... More
Many long-standing image processing problems in applied science domains are finding solutions through the application of deep learning approaches to image processing. Here we present one such application; the case of classifying images of protein crystallisation droplets. The Collaborative Crystallisation Centre in Melbourne, Australia is a medium throughput service facility that produces between five and twenty thousand images per day. This submission outlines a reliable and robust machine learning pipeline that autonomously classifies these images using CSIRO’s high-performance computing facilities. Our pipeline achieves improved accuracies over existing implementations and delivers these results in real time. We discuss the specific tools and techniques used to construct the pipeline, as well as the methodologies for testing and validating externally developed classification models. Less
The three members of the endocrine fibroblast growth factor FGF family designated FGF FGF and FGF mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor FGFR bound to either -Klotho or -Klotho receptors Structural analyses of ligand-occupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF and FGF to -Klotho or -Klotho respectively They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling Here we describe the crystal structure the C-terminal tail of ... More
The three members of the endocrine fibroblast growth factor (FGF) family designated FGF19, FGF21, and FGF23 mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor (FGFR) bound to either α-Klotho or β-Klotho receptors. Structural analyses of ligand-occupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF21 and FGF23 to β-Klotho or α-Klotho, respectively. They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling. Here we describe the crystal structure the C-terminal tail of FGF19 (FGF19CT) bound to sKLB and demonstrate that FGF19CT and FGF21CT bind to the same binding site on sKLB, via a multiturn D-P motif to site 1 and via a S-P-S motif to the pseudoglycoside hydrolase region (site 2). Binding affinities to sKLB and cellular stimulatory activities of FGF19CT, FGF21CT, and a variety of chimeric mutants to cells expressing β-Klotho together with FGFR1c or FGFR4 were also analyzed. These experiments as well as detailed comparison of the structures of free and ligand-occupied sKLB to the structure of ligand-occupied sKLA reveal a general mechanism for recognition of endocrine FGFs by Klotho proteins and regulatory interactions with FGFRs that control their pleiotropic cellular responses. 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
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
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
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
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
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
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
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
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
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
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
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
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
The bacterium Streptococcus pneumoniae the pneumococcus is a major human pathogen that requires Zn for its survival and virulence in the host environment Polyhistidine triad protein D PhtD has a known role in pneumococcal Zn homeostasis However the mechanistic basis of PhtD function remains unclear partly due to a lack of structural information Here we determined the crystal structure of the fragment PhtD - containing the third Zn -binding histidine triad HT motif of the protein Analysis of the structure suggests that Zn binding occurs at the surface of the protein and that all five HT motifs in the protein ... More
The bacterium Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that requires Zn2+ for its survival and virulence in the host environment. Polyhistidine triad protein D (PhtD) has a known role in pneumococcal Zn2+ homeostasis. However, the mechanistic basis of PhtD function remains unclear, partly due to a lack of structural information. Here, we determined the crystal structure of the fragment PhtD269-339 , containing the third Zn2+ -binding histidine triad (HT) motif of the protein. Analysis of the structure suggests that Zn2+ binding occurs at the surface of the protein and that all five HT motifs in the protein bind Zn2+ and share similar structures. These new structural insights aid in our understanding of how the Pht proteins facilitate pneumococcal Zn2+ acquisition. Less
Type-A -aminobutyric acid GABAA receptors are pentameric ligand-gated ion channels pLGICs typically consisting of subunit combinations They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs such as benzodiazepines BZDs used to treat epilepsy insomnia anxiety panic disorder and muscle spasm However the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown Here we report the cryo-EM structure of a human GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator PAM The receptor subunits assume a unified quaternary activated conformation ... More
Type-A γ-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of α/β/γ subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human α1β3γ2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered α5 and α5γ2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation. Less
Platelet-activating-factor receptor PAFR responds to platelet-activating factor PAF a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects PAFR is considered an important drug target for treating asthma inflammation and cardiovascular diseases Here we report crystal structures of human PAFR in complex with the antagonist SR and the inverse agonist ABT- at - and - resolution respectively The structures supported by molecular docking of PAF provide insights into the signal-recognition mechanisms of PAFR The PAFR SR structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by and respectively and helix VIII ... More
Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR–SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors. Less
MicroED structure of the NaK ion channel reveals a Na+ partition process into the selectivity filter
Sodium Na is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation signaling and facilitation of various transporters The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels yet they are rarely captured by conventional structural biology means Here we use the emerging cryo-electron microscopy cryoEM method micro-electron diffraction MicroED to study the structure of a prototypical tetrameric Na -conducting channel NaK to resolution from nano-crystals Two new conformations at the external site of NaK are identified allowing us to visualize a partially ... More
Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics. Less
Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates Solution conditions affect morphologic features such as crystal size as well as kinetic features such as crystal growth time Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols These techniques have limitations in terms of handling large data sets comprehensiveness or completeness To eliminate these limitations morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have ... More
Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphologic features, such as crystal size, as well as kinetic features, such as crystal growth time. Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols. These techniques have limitations in terms of handling large data sets, comprehensiveness or completeness. To eliminate these limitations, morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram method. Morphologic features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength, and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the empirical phase diagram method can support high-throughput crystallization experiments in its data amount as well as its data complexity. Less
Neuropeptide Y NPY receptors belong to the G protein-coupled receptor GPCR superfamily and play important roles in food intake anxiety and cancer regulation The NPY Y receptor system has emerged as one of the most complex networks with three peptide ligands NPY peptide YY and pancreatic polypeptide binding to four receptors in mammals namely Y Y Y and Y receptors with different affinity and selectivity NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y receptor Y R A number of peptides and small-molecule compounds have been characterized as Y R antagonists and ... More
Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. The NPY/Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in mammals, namely Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumor1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors. Less
As a protective envelope surrounding the bacterial cell the peptidoglycan sacculus is a site of vulnerability and an antibiotic target Peptidoglycan components assembled in the cytoplasm are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate A product of peptidoglycan synthesis undecaprenyl-pyrophosphate is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase BacA To understand how BacA functions we determine its crystal structure at resolution The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane Conserved residues map to the pocket where pyrophosphorolysis occurs BacA ... More
As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase. Less
Investigation of the biochemical basis of life is a long standing challenge for scientists The field of molecular biology was established based on the pioneering studies of macromolecular structures including the structural characterization of the DNA double helix and the description of the first protein structures These early achievements along with the technical development e g automatic data collection and computer-assisted data analysis enables extremely efficient and fast determination of structure and dynamic properties of proteins
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the -oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA RNA production This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring To provide validation for this approach as a drug target we have RNAi silenced the three -oxopurine phosphoribosyltransferase PRTase isoforms in the infectious stage of Trypanosoma brucei demonstrating that ... More
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery. Less
Plastidial thioredoxin TRX -like proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs we solved the crystal structures of poplar TRX-like in oxidized and reduced states and of mutated variants These structures share similar folding with TRXs exhibiting the canonical WCGPC signature Moreover the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C S C S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins Modeling of the ... More
Plastidial thioredoxin (TRX)-like2.1 proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling. To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs, we solved the crystal structures of poplar TRX-like2.1 in oxidized and reduced states and of mutated variants. These structures share similar folding with TRXs exhibiting the canonical WCGPC signature. Moreover, the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C45S/C67S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins. Modeling of the interaction of TRX-like2.1 with both NADPH- and ferredoxin-thioredoxin reductases (FTR) indicates that the presence of Arg43 and Lys44 residues likely precludes reduction by the plastidial FTR. Less
The ageing suppressor -klotho binds to the fibroblast growth factor receptor FGFR This commits FGFR to respond to FGF a key hormone in the regulation of mineral ion and vitamin D homeostasis The role and mechanism of this co-receptor are unknown Here we present the atomic structure of a ternary complex that consists of the shed extracellular domain of -klotho the FGFR c ligand-binding domain and FGF In this complex -klotho simultaneously tethers FGFR c by its D domain and FGF by its C-terminal tail thus implementing FGF FGFR c proximity and conferring stability Dimerization of the stabilized ternary complexes ... More
The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23–FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling. Less
Canonical FGFs activate FGF receptors FGFR via paracrine or autocrine mechanisms in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation By contrast endocrine FGFs FGF and are circulating hormones that regulate critical metabolic processes in a variety of tissues FGF regulates bile acid synthesis and lipogenesis while FGF stimulate insulin sensitivity energy expenditure and weight loss Endocrine FGFs signal through FGFRs in a manner that requires Klothos which are cell surface proteins with tandem glycosidase domains Here we describe the crystal structures of free and ligand-bound -Klotho extracellular regions revealing the molecular mechanism ... More
Canonical FGFs activate FGF receptors (FGFR) via paracrine or autocrine mechanisms, in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation1,2. By contrast, endocrine FGFs (FGF 19, 21, and 23) are circulating hormones that regulate critical metabolic processes in a variety of tissues3,4. FGF19 regulates bile acid synthesis and lipogenesis, while FGF21 stimulate insulin sensitivity, energy expenditure and weight loss5. Endocrine FGFs signal through FGFRs in a manner that requires Klothos, which are cell surface proteins with tandem glycosidase domains3,4. Here we describe the crystal structures of free and ligand-bound β-Klotho extracellular regions, revealing the molecular mechanism underlying the specificity of FGF21 towards β-Klotho and demonstrating how FGFR is activated in a Klotho-dependent manner. β-Klotho serves as a primary “Zip code”-like receptor for FGF21 with an FGFR functioning as a catalytic subunit that mediates intracellular signaling. Our structures also show how a sugar cutting enzyme (glycosidase) has evolved to become a specific receptor for hormones that regulate metabolic processes including lowering of blood sugar. Finally, we describe a superior agonistic variant of FGF21 and present structural insights offering development of novel therapeutics for diseases linked to endocrine FGFs. Less
The maturation of Ras GTPases and other cellular CaaX proteins involves three enzymatic steps addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine C in the C-terminal CaaX motif proteolytic cleavage of the aaX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase ICMT a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum ER ICMT is the only cellular enzyme known to methylate prenylcysteine substrates methylation is important for their biological functions including the membrane localisations and subsequent activities of Ras prelamin A and Rab ICMT ... More
The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes. Less
Class B G-protein-coupled receptors GPCRs which consist of an extracellular domain ECD and a transmembrane domain TMD respond to secretin peptides to play a key part in hormonal homeostasis and are important therapeutic targets for a variety of diseases Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket Recently three structures of class B GPCRs in complex with peptide ligands have been solved These structures provide essential ... More
Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases1,2,3,4,5,6,7,8. Previous work9,10,11 has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved12,13,14. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs. Less
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task However by a systematic screening approach the time to obtain suitable ... More
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties. Less
Ferredoxin-dependent bilin reductases FDBRs are a class of enzymes reducing the heme metabolite biliverdin IX BV to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms Thus far seven FDBR families have been identified each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate The newest addition to the family is PcyX originally identified from metagenomics data derived from phage Phylogenetically PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin PcyX however converts the same substrate to phycoerythrobilin resembling the reaction catalysed by cyanophage PebS Within this study ... More
Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Thus far, seven FDBR families have been identified, each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate. The newest addition to the family is PcyX, originally identified from metagenomics data derived from phage. Phylogenetically, PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin. PcyX, however, converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. Within this study, we aimed at understanding the evolution of catalytic activities within FDBRs using PcyX as an example. Additional members of the PcyX clade and a remote member of the PcyA family were investigated to gain insights into catalysis. Biochemical data in combination with the PcyX crystal structure revealed that a conserved aspartate-histidine pair is critical for activity. Interestingly, the same residues are part of a catalytic Asp-His-Glu triad in PcyA, including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu failed to convert PcyX to a PcyA. Therefore, the change in regiospecificity is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products. Less