The Mycobacterium tuberculosis Ser/Thr kinase substrate Rv2175c is a DNA-binding protein regulated by phosphorylation
| Autor: | Christian Roumestand, Charlotte Stagier-Simon, Laurent Kremer, Marc J. Canova, Virginie Molle, Martin Cohen-Gonsaud, Philippe Barthe |
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| Přispěvatelé: | Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Dynamique des interactions membranaires normales et pathologiques (DIMNP), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Bosch-Savary, Annie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université Montpellier 1 (UM1) |
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Magnetic Resonance Spectroscopy MESH: Mycobacterium tuberculosis MESH: Sequence Homology Amino Acid [SDV]Life Sciences [q-bio] MESH: Protein Structure Secondary Electrophoretic Mobility Shift Assay MESH: Amino Acid Sequence Biochemistry Protein Structure Secondary Substrate Specificity Serine MESH: Protein Structure Tertiary Protein phosphorylation Cloning Molecular Phosphorylation Threonine MESH: Bacterial Proteins ComputingMilieux_MISCELLANEOUS 0303 health sciences Kinase Protein Synthesis Post-Translational Modification and Degradation 3. Good health DNA-Binding Proteins [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology MESH: Models Molecular Protein Binding MESH: Mutation Molecular Sequence Data Fluorescence Polarization Protein Serine-Threonine Kinases Biology MESH: Protein-Serine-Threonine Kinases 03 medical and health sciences Bacterial Proteins [SDV.BBM] Life Sciences [q-bio]/Biochemistry Molecular Biology MESH: Protein Binding Electrophoretic mobility shift assay MESH: Cloning Molecular [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Amino Acid Sequence Molecular Biology 030304 developmental biology MESH: Fluorescence Polarization Binding Sites Protein-Serine-Threonine Kinases MESH: Molecular Sequence Data Sequence Homology Amino Acid MESH: Phosphorylation 030306 microbiology MESH: Magnetic Resonance Spectroscopy Binding protein Mycobacterium tuberculosis Cell Biology [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology Protein Structure Tertiary MESH: Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization MESH: Binding Sites Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutation MESH: Electrophoretic Mobility Shift Assay MESH: Substrate Specificity MESH: Chromatography Liquid MESH: DNA-Binding Proteins Chromatography Liquid |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2009, 284 (29), pp.19290-19300. ⟨10.1074/jbc.M109.019653⟩ Journal of Biological Chemistry, 2009, 284 (29), pp.19290-19300. ⟨10.1074/jbc.M109.019653⟩ |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.M109.019653⟩ |
| Popis: | International audience; Recent efforts have underlined the role of serine/threonine protein kinases in growth, pathogenesis, and cell wall metabolism in Mycobacterium tuberculosis. Although most kinases have been investigated for their physiological roles, little information is available regarding how serine/threonine protein kinase-dependent phosphorylation regulates the activity of kinase substrates. Herein, we focused on M. tuberculosis Rv2175c, a protein of unknown function, conserved in actinomycetes, and recently identified as a substrate of the PknL kinase. We solved the solution structure of Rv2175c by multidimensional NMR and demonstrated that it possesses an original winged helix-turn-helix motif, indicative of a DNA-binding protein. The DNA-binding activity of Rv2175c was subsequently confirmed by fluorescence anisotropy, as well as in electrophoretic mobility shift assays. Mass spectrometry analyses using a combination of MALDI-TOF and LC-ESI/MS/MS identified Thr(9) as the unique phosphoacceptor. This was further supported by complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. Importantly, the DNA-binding activity was completely abrogated in a Rv2175c_T9D mutant, designed to mimic constitutive phosphorylation, but not in a mutant lacking the first 13 residues. This implies that the function of the N-terminal extension is to provide a phosphoacceptor (Thr(9)), which, following phosphorylation, negatively regulates the Rv2175c DNA-binding activity. Interestingly, the N-terminal disordered extension, which bears the phosphoacceptor, was found to be restricted to members of the M. tuberculosis complex, thus suggesting the existence of an original mechanism that appears to be unique to the M. tuberculosis complex. |
| Databáze: | OpenAIRE |
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