Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling.

Autor:	Wagner T; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., André-Leroux G; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., Hindie V; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., Barilone N; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., Lisa MN; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., Hoos S; Institut Pasteur, Plateforme de Biophysique Moléculaire, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., Raynal B; Institut Pasteur, Plateforme de Biophysique Moléculaire, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., Vulliez-Le Normand B; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France., O'Hare HM; Leicester Tuberculosis Research Group (LTBRG) and Leicester Institute of Structural and Chemical Biology (LISCB), Department of Respiratory Science & Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 7RH, UK., Bellinzoni M; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. pedro.alzari@pasteur.fr marco.bellinzoni@pasteur.fr., Alzari PM; Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 & Université Paris Diderot, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. pedro.alzari@pasteur.fr marco.bellinzoni@pasteur.fr.
Jazyk:	angličtina
Zdroj:	Science signaling [Sci Signal] 2019 May 07; Vol. 12 (580). Date of Electronic Publication: 2019 May 07.
DOI:	10.1126/scisignal.aav9504
Abstrakt:	Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA-binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners. (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
Databáze:	MEDLINE
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