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ABSTRACT: Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1-2 and a deletion near site Ca3.
Acta crystallographica. Section D, Biological crystallography 01/2013; 69(Pt 1):24-31. · 12.67 Impact Factor
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Leo A Hardegger,
Bernd Kuhn,
Beat Spinnler,
Lilli Anselm,
Robert Ecabert, Martine Stihle,
Bernard Gsell,
Ralf Thoma,
Joachim Diez,
Jörg Benz,
Jean-Marc Plancher,
Guido Hartmann,
Yoshiaki Isshiki,
Kenji Morikami,
Nobuo Shimma,
Wolfgang Haap,
David W Banner,
François Diederich
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ABSTRACT: In two series of small-molecule ligands, one inhibiting human cathepsin L (hcatL) and the other MEK1 kinase, biological affinities were found to strongly increase when an aryl ring of the inhibitors is substituted with the larger halogens Cl, Br, and I, but to decrease upon F substitution. X-ray co-crystal structure analyses revealed that the higher halides engage in halogen bonding (XB) with a backbone C=O in the S3 pocket of hcatL and in a back pocket of MEK1. While the S3 pocket is located at the surface of the enzyme, which provides a polar environment, the back pocket in MEK1 is deeply buried in the protein and is of pronounced apolar character. This study analyzes environmental effects on XB in protein-ligand complexes. It is hypothesized that energetic gains by XB are predominantly not due to water replacements but originate from direct interactions between the XB donor (Caryl-X) and the XB acceptor (C=O) in the correct geometry. New X-ray co-crystal structures in the same crystal form (space group P2(1)2(1)2(1)) were obtained for aryl chloride, bromide, and iodide ligands bound to hcatL. These high-resolution structures reveal that the backbone C=O group of Gly61 in most hcatL co-crystal structures maintains water solvation while engaging in XB. An aryl-CF3-substituted ligand of hcatL with an unexpectedly high affinity was found to adopt the same binding geometry as the aryl halides, with the CF3 group pointing to the C=O group of Gly61 in the S3 pocket. In this case, a repulsive F2C-F⋅⋅⋅O=C contact apparently is energetically overcompensated by other favorable protein-ligand contacts established by the CF3 group.
ChemMedChem 09/2011; 6(11):2048-54. · 3.15 Impact Factor
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Claudia Ferrara,
Sandra Grau,
Christiane Jäger,
Peter Sondermann,
Peter Brünker,
Inja Waldhauer,
Michael Hennig,
Armin Ruf,
Arne Christian Rufer, Martine Stihle,
Pablo Umaña,
Jörg Benz
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ABSTRACT: Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen–antibody
complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity
for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next
generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In
this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were
determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in
improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate–carbohydrate interactions between
glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts
are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the
higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune
system can regulate antibody-mediated effector functions.
Proceedings of the National Academy of Sciences 08/2011; 108(31):12669-12674. · 9.68 Impact Factor
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ABSTRACT: E-ISA247 (voclosporin) is a cyclosporin A analogue that is in late-stage clinical development for the treatment of autoimmune diseases and the prevention of organ graft rejection. The X-ray crystal structures of E-ISA247 and its stereoisomer Z-ISA247 bound to cyclophilin A have been determined and their binding affinities were measured to be 15 and 61 nM, respectively, by fluorescence spectroscopy. The higher affinity of E-ISA247 can be explained by superior van der Waals contacts between its unique side chain and cyclophilin A. Comparison with the known ternary structure including calcineurin suggests that the higher immunosuppressive efficacy of E-ISA247 relative to cyclosporin A could be a consequence of structural changes in calcineurin induced by the modified E-ISA247 side chain.
Acta crystallographica. Section D, Biological crystallography 02/2011; 67(Pt 2):119-23. · 12.67 Impact Factor
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Leo A Hardegger,
Bernd Kuhn,
Beat Spinnler,
Lilli Anselm,
Robert Ecabert, Martine Stihle,
Bernard Gsell,
Ralf Thoma,
Joachim Diez,
Jörg Benz,
Jean-Marc Plancher,
Guido Hartmann,
David W Banner,
Wolfgang Haap,
François Diederich
Angewandte Chemie International Edition 01/2011; 50(1):314-8. · 13.45 Impact Factor
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ABSTRACT: The human glucocorticoid receptor ligand-binding domain (hGR-LBD) is an important drug target for the treatment of various diseases. However, the low intrinsic stability and solubility of hGR-LBD have rendered its purification and biophysical characterization difficult. In order to overcome these problems, we have stabilized hGR-LBD by a combination of random mutagenesis and high-throughput screening using fluorescence-activated cell sorting (FACS) with enhanced green fluorescent protein (eGFP) as folding reporter. Two plasmid-encoded gene libraries of hGR-LBD fused to the egfp gene were expressed in Escherichia coli, followed by eight rounds of FACS screening, in each of which 10(8) cells were analyzed. The hgr-lbd mutants isolated by this approach contained numerous amino acid exchanges, and four beneficial ones (A605V, V702A, E705G, and M752T) were followed up in detail. Their characterization showed that the fluorescence of hGR-LBD-eGFP fusions is correlated linearly with the stability and solubility of hGR-LBD in the absence of eGFP. When combined, the four exchanges increased the thermal stability of hGR-LBD by more than 8 °C and enhanced its purification yield after expression in E. coli by about 26-fold. The introduction of three beneficial exchanges into the homologous ligand-binding domain of mouse enabled its X-ray structure determination at high resolution, which showed how the exchanges stabilize the protein and revealed atomic details that will guide future drug design. Our results demonstrate that large eGFP fusion libraries can be screened by FACS with extreme sensitivity and efficiency, yielding stabilized eukaryotic proteins suitable for biophysical characterization and structure determination.
Journal of Molecular Biology 11/2010; 403(4):562-77. · 4.00 Impact Factor
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ABSTRACT: The human fatty acid synthase (FAS) is a key enzyme in the metabolism of fatty acids and a target for antineoplastic and antiobesity drug development. Due to its size and flexibility, structural studies of mammalian FAS have been limited to individual domains or intermediate-resolution studies of the complete porcine FAS. We describe the high-resolution crystal structure of a large part of human FAS that encompasses the tandem domain of beta-ketoacyl synthase (KS) connected by a linker domain to the malonyltransferase (MAT) domain. Hinge regions that allow for substantial flexibility of the subdomains are defined. The KS domain forms the canonical dimer, and its substrate-binding site geometry differs markedly from that of bacterial homologues but is similar to that of the porcine orthologue. The didomain structure reveals a possible way to generate a small and compact KS domain by omitting a large part of the linker and MAT domains, which could greatly aid in rapid screening of KS inhibitors. In the crystal, the MAT domain exhibits two closed conformations that differ significantly by rigid-body plasticity. This flexibility may be important for catalysis and extends the conformational space previously known for type I FAS and 6-deoxyerythronolide B synthase.
Journal of Molecular Biology 03/2010; 397(2):508-19. · 4.00 Impact Factor
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ABSTRACT: Mifepristone is known to induce mixed passive antagonist, active antagonist, and agonist effects via the glucocorticoid receptor (GR) pathway. Part of the antagonist effects of mifepristone are due to the repression of gene transcription mediated by the nuclear receptor corepressor (NCoR). Here, we report the crystal structure of a ternary complex of the GR ligand binding domain (GR-LBD) with mifepristone and a receptor-interacting motif of NCoR. The structures of three different conformations of the GR-LBD mifepristone complex show in the oxosteroid hormone receptor family how helix 12 modulates LBD corepressor and coactivator binding. Differences in NCoR binding and in helix 12 conformation reveal how the 11beta substituent in mifepristone triggers the helix 12 molecular switch to reshape the coactivator site into the corepressor site. Two observed conformations exemplify the active antagonist state of GR with NCoR bound. In another conformation, helix 12 completely blocks the coregulator binding site and explains the passive antagonistic effect of mifepristone on GR.
Journal of Molecular Biology 11/2009; 395(3):568-77. · 4.00 Impact Factor
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Andreas Kuglstatter,
Martin Stahl,
Jens-Uwe Peters,
Walter Huber, Martine Stihle,
Daniel Schlatter,
Jörg Benz,
Armin Ruf,
Doris Roth,
Thilo Enderle,
Michael Hennig
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ABSTRACT: Fragment screening revealed that tyramine binds to the active site of the Alzheimer's disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.
Bioorganic & medicinal chemistry letters 03/2008; 18(4):1304-7. · 2.65 Impact Factor
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Elke Burgermeister,
Astride Schnoebelen,
Angele Flament,
Jörg Benz, Martine Stihle,
Bernard Gsell,
Arne Rufer,
Armin Ruf,
Bernd Kuhn,
Hans Peter Märki,
Jacques Mizrahi,
Elena Sebokova,
Eric Niesor,
Markus Meyer
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ABSTRACT: Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.
Molecular Endocrinology 05/2006; 20(4):809-30. · 4.54 Impact Factor
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ABSTRACT: Carnitine palmitoyltransferases 1 and 2 (CPTs) facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. We report here the 1.6 A resolution structure of the full-length mitochondrial membrane protein CPT-2. The structure of CPT-2 in complex with the generic CPT inhibitor ST1326 ([R]-N-[tetradecylcarbamoyl]-aminocarnitine), a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for diabetes mellitus treatment, was solved at 2.5 A resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of antidiabetic drugs. We identify a sequence insertion found in CPT-2 that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2.
Structure 05/2006; 14(4):713-23. · 6.35 Impact Factor
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Angewandte Chemie International Edition 08/2005; 44(28):4400-4. · 13.45 Impact Factor
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ABSTRACT: In the pharmaceutical industry, knowledge of the three-dimensional structure of a specific target facilitates the drug-discovery process. Despite possessing favoured analytical properties such as high purity and monodispersion in light scattering, some proteins are not capable of forming crystals suitable for X-ray analysis. Cyclophilin D, an isoform of cyclophilin that is expressed in the mitochondria, was selected as a drug target for the treatment of cardiac disorders. As the wild-type enzyme defied all attempts at crystallization, protein engineering on the enzyme surface was performed. The K133I mutant gave crystals that diffracted to 1.7 A resolution using in-house X-ray facilities and were suitable for soaking experiments. The crystals were very robust and diffraction was maintained after soaking in 25% DMSO solution: excellent conditions for the rapid analysis of complex structures including crystallographic fragment screening.
Acta Crystallographica Section D Biological Crystallography 06/2005; 61(Pt 5):513-9. · 12.62 Impact Factor
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ABSTRACT: In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.
Nature 12/2004; 432(7013):118-22. · 36.28 Impact Factor
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ABSTRACT: The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC(50) for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.
Biochemical and Biophysical Research Communications 04/2004; 315(2):247-54. · 2.48 Impact Factor
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ABSTRACT: Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.
Structure 09/2003; 11(8):947-59. · 6.35 Impact Factor
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Angewandte Chemie International Edition 07/2003; 42(22):2507-11. · 13.45 Impact Factor
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ABSTRACT: In this study, characterization and optimization of a modified microbatch crystallization technique has been attempted in order to provide a rapid screening method. Using this method for screening has certain advantages over standard vapour-diffusion methods: no sealing of drops is required, no reservoir solutions are needed and the experiments can easily be performed over a range of temperatures.
Acta Crystallographica Section D Biological Crystallography 03/2003; 59(Pt 2):396-9. · 12.62 Impact Factor
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ABSTRACT: The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC50 for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.
Biochemical and Biophysical Research Communications.
