22 research outputs found
New insights into the enzymatic mechanism of human chitotriosidase (CHIT1) catalytic domain by atomic resolution X-ray diffraction and hybrid QM/MM
Chitotriosidase (CHIT1) is a human chitinase belonging to the highly conserved glycosyl hydrolase family 18 (GH18). GH18 enzymes hydrolyze chitin, an N-acetylglucosamine polymer synthesized by lower organisms for structural purposes. Recently, CHIT1 has attracted attention owing to its upregulation in immune-system disorders and as a marker of Gaucher disease. The 39 kDa catalytic domain shows a conserved cluster of three acidic residues, Glu140, Asp138 and Asp136, involved in the hydrolysis reaction. Under an excess concentration of substrate, CHIT1 and other homologues perform an additional activity, transglycosylation. To understand the catalytic mechanism of GH18 chitinases and the dual enzymatic activity, the structure and mechanism of CHIT1 were analyzed in detail. The resolution of the crystals of the catalytic domain was improved from 1.65 Ă
(PDB entry 1waw ) to 0.95-1.10 Ă
for the apo and pseudo-apo forms and the complex with chitobiose, allowing the determination of the protonation states within the active site. This information was extended by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. The results suggest a new mechanism involving changes in the conformation and protonation state of the catalytic triad, as well as a new role for Tyr27, providing new insights into the hydrolysis and transglycosylation activities.Fil: Fadel, Firas. Centre National de la Recherche Scientifique; Francia. Institut de GĂ©nĂ©tique et de Biologie MolĂ©culaire et Cellulaire; FranciaFil: Zhao, Yuguang. University of Oxford; Reino UnidoFil: Cachau, Raul. Frederick National Laboratory for Cancer Research; Estados UnidosFil: Cousido Siah, Alexandra. Centre National de la Recherche Scientifique; Francia. Institut de GĂ©nĂ©tique et de Biologie MolĂ©culaire et Cellulaire; FranciaFil: Ruiz, Francesc X.. Centre National de la Recherche Scientifique; Francia. Institut de GĂ©nĂ©tique et de Biologie MolĂ©culaire et Cellulaire; FranciaFil: Harlos, Karl. University of Oxford; Reino UnidoFil: Howard, Eduardo Ignacio. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - La Plata. Instituto de FĂsica de LĂquidos y Sistemas BiolĂłgicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de FĂsica de LĂquidos y Sistemas BiolĂłgicos; Argentina. Centre National de la Recherche Scientifique; Francia. Institut de GĂ©nĂ©tique et de Biologie MolĂ©culaire et Cellulaire; FranciaFil: Mitschler, Andre. Centre National de la Recherche Scientifique; Francia. Institut de GĂ©nĂ©tique et de Biologie MolĂ©culaire et Cellulaire; FranciaFil: Podjarny, Alberto Daniel. Centre National de la Recherche Scientifique; Francia. Institut de GĂ©nĂ©tique et de Biologie MolĂ©culaire et Cellulaire; Franci
New insights into the enzymatic mechanism of human chitotriosidase (CHIT1) catalytic domain by atomic resolution X-ray diffraction and hybrid QM/MM
Chitotriosidase (CHIT1) is a human chitinase belonging to the highly conserved glycosyl hydrolase family 18 (GH18). GH18 enzymes hydrolyze chitin, an N-acetylglucosamine polymer synthesized by lower organisms for structural purposes. Recently, CHIT1 has attracted attention owing to its upregulation in immune-system disorders and as a marker of Gaucher disease. The 39 kDa catalytic domain shows a conserved cluster of three acidic residues, Glu140, Asp138 and Asp136, involved in the hydrolysis reaction. Under an excess concentration of substrate, CHIT1 and other homologues perform an additional activity, transglycosylation. To understand the catalytic mechanism of GH18 chitinases and the dual enzymatic activity, the structure and mechanism of CHIT1 were analyzed in detail. The resolution of the crystals of the catalytic domain was improved from 1.65 Ă
(PDB entry 1waw ) to 0.95-1.10 Ă
for the apo and pseudo-apo forms and the complex with chitobiose, allowing the determination of the protonation states within the active site. This information was extended by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. The results suggest a new mechanism involving changes in the conformation and protonation state of the catalytic triad, as well as a new role for Tyr27, providing new insights into the hydrolysis and transglycosylation activities.Instituto de FĂsica de LĂquidos y Sistemas BiolĂłgico
Discovery of (R)-2-Amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic Acid and Congeners As Highly Potent Inhibitors of Human Arginases I and II for Treatment of Myocardial Reperfusion Injury
Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structureâactivity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.Fil: Van Zandt, Michael C.. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Whitehouse, Darren L.. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Golebiowski, Adam. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Ji, Min Koo. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Zhang, Mingbao. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Beckett, R. Paul. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Jagdmann, G. Erik. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Ryder, Todd R.. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Sheeler, Ryan. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Andreoli, Monica. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Conway, Bruce. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Mahboubi, Keyvan. Institutes for Pharmaceutical Discovery; Estados UnidosFil: DâAngelo, Gerard. Institutes for Pharmaceutical Discovery; Estados UnidosFil: Mitschler, Andre. UniversitĂ© de Strasbourg; FranciaFil: Cousido Siah, Alexandra. UniversitĂ© de Strasbourg; FranciaFil: Ruiz, Frances X.. UniversitĂ© de Strasbourg; FranciaFil: Howard, Eduardo Ignacio. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - La Plata. Instituto de FĂsica de LĂquidos y Sistemas BiolĂłgicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de FĂsica de LĂquidos y Sistemas BiolĂłgicos; Argentina. UniversitĂ© de Strasbourg; FranciaFil: Podjarny, Alberto Daniel. UniversitĂ© de Strasbourg; FranciaFil: Schroeter, Hagen. Mars Incorporated; Estados Unido
Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53
The p53 pro-apoptotic tumor suppressor is mutated or functionally altered in most cancers. In epithelial tumors induced by âhigh-riskâ mucosal Human Papillomaviruses (hrm-HPVs), including human cervical carcinoma and a growing number of head-and-neck cancers (1), p53 is degraded by the viral oncoprotein E6 (2). In this process, E6 binds to a short LxxLL consensus sequence within the cellular ubiquitin ligase E6AP (3). Subsequently, the E6/E6AP heterodimer recruits and degrades p53 (4). Neither E6 nor E6AP are separately able to recruit p53 (3,5), and the precise mode of assembly of E6, E6AP and p53 is unknown. Here, we solved the crystal structure of a ternary complex comprising full-length HPV16 E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6-p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumor suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against HPV-mediated oncogenesis
Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants
Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.PMC529941
Preparation of 3,3',4,4'-tetramethyl-1,1'-biphospholyl and its reactions with iron and cobalt carbonyls. Synthesis and x-ray crystal structure of a phospholyl complex with a chain of four cobalt atoms
Reaction of lithium alkyls and aryls with 1,1'-diphosphaferrocenes. Synthesis and structure of a stable bis(diene)iron(-I) species
Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein
Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85â
Ă
resolution from a 0.13â
mm3 crystal