scholarly journals Structural framework of c-Src activation by integrin β3

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 700-706 ◽  
Author(s):  
Run Xiao ◽  
Xiao-Dong Xi ◽  
Zhu Chen ◽  
Sai-Juan Chen ◽  
Guoyu Meng
Keyword(s):  
Crystal Structure ◽  
Tumor Cells ◽  
Structural Information ◽  
Intramolecular Interaction ◽  
Sh3 Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray ◽  
Integrin Β3 ◽  
Structural Framework

Abstract The integrin β3-mediated c-Src priming and activation, via the SH3 domain, is consistently associated with diseases, such as the formation of thrombosis and the migration of tumor cells. Conventionally, activation of c-Src is often induced by the binding of proline-rich sequences to its SH3 domain. Instead, integrin β3 uses R760GT762 for priming and activation. Because of the lack of structural information, it is not clear where RGT will bind to SH3, and under what mechanism this interaction can prime/activate c-Src. In this study, we present a 2.0-Å x-ray crystal structure in which SH3 is complexed with the RGT peptide. The binding site lies in the “N”-Src loop of the SH3 domain. Structure-based site-directed mutagenesis showed that perturbation on the “N”-Src loop disrupts the interaction between the SH3 domain and the RGT peptide. Furthermore, the simulated c-Src:β3 complex based on the crystal structure of SH3:RGT suggests that the binding of the RGT peptide might disrupt the intramolecular interaction between the SH3 and linker domains, leading to the disengagement of Trp260:“C”-helix and further activation of c-Src.

The X-ray Crystal Structure of the First RNA Recognition Motif and Site-Directed Mutagenesis Suggest a Possible HuR Redox Sensing Mechanism

2010 ◽  
Vol 397 (5) ◽  
pp. 1231-1244 ◽  
Author(s):  
Roger Marc Benoit ◽  
Nicole-Claudia Meisner ◽  
Joerg Kallen ◽  
Patrick Graff ◽  
René Hemmig ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
X Ray ◽  
Sensing Mechanism ◽  
Recognition Motif ◽  
Redox Sensing

scholarly journals Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwen Liu ◽  
Fanglong Zhao ◽  
Boyang Zhao ◽  
Jie Yang ◽  
Joseph Ferrara ◽  
...  
Keyword(s):  
High Resolution ◽  
Organic Synthesis ◽  
Indole Alkaloids ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Computational Studies ◽  
X Ray ◽  
Evolutionary Branch ◽  
Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

scholarly journals X-Ray Structure and Site-Directed Mutagenesis Analysis of the Escherichia coli Colicin M Immunity Protein

2010 ◽  
Vol 193 (1) ◽  
pp. 205-214 ◽  
Author(s):  
F. Gerard ◽  
M. A. Brooks ◽  
H. Barreteau ◽  
T. Touze ◽  
M. Graille ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Immunity Protein ◽  
X Ray ◽  
Colicin M

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

scholarly journals Crystal Structure and Site-directed Mutagenesis ofBacillus maceransEndo-1,31,4--glucanase

1995 ◽  
Vol 270 (7) ◽  
pp. 3081-3088 ◽  
Author(s):  
Michael Hahn ◽  
Ole Olsen ◽  
Oliver Politz ◽  
Rainer Borriss ◽  
Udo Heinemann
Keyword(s):  
Crystal Structure ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Crystal Structure and Site-directed Mutagenesis of Enzymatic Components from Clostridium perfringens Iota-toxin

2003 ◽  
Vol 325 (3) ◽  
pp. 471-483 ◽  
Author(s):  
Hideaki Tsuge ◽  
Masahiro Nagahama ◽  
Hiroyuki Nishimura ◽  
Junzo Hisatsune ◽  
Yoshihiko Sakaguchi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Clostridium Perfringens ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Clostridium Perfringens Iota Toxin

Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Alisson C Cardoso ◽  
Ana H Pereira ◽  
Andre L Ambrosio ◽  
Silvio R Consonni ◽  
Sandra M Dias ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mechanical Stress ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Mechanistic Model ◽  
Focal Adhesions ◽  
Saxs Data ◽  
X Ray ◽  
Gene Assay

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

scholarly journals Conformational communication mediates the reset step in t6A biosynthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6551-6567 ◽  
Author(s):  
Amit Luthra ◽  
Naduni Paranagama ◽  
William Swinehart ◽  
Susan Bayooz ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translational Fidelity ◽  
Substrate Analog ◽  
Binding Cavity ◽  
Saxs Analysis

Abstract 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.

scholarly journals Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Gongquan Liu ◽  
Weiwei Wang ◽  
Fangyuan He ◽  
Peng Zhang ◽  
Ping Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
Amine Oxidase ◽  
Substrate Binding ◽  
Docking Study ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Structure Comparison ◽  
Content Type ◽  
Key Enzyme

ABSTRACT Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a “butterfly bend” conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products. IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

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