combined pdf.pdf (18.26 MB)
Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling.
journal contribution
posted on 2019-05-13, 13:35 authored by T Wagner, G André-Leroux, V Hindie, N Barilone, M-N Lisa, S Hoos, B Raynal, B Vulliez-Le Normand, HM O'Hare, M Bellinzoni, PM AlzariForkhead-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.
Funding
This work was partially supported by grants from the Institut Pasteur, the CNRS, and the European Commission’s Seventh Framework Programme (MM4TB, grant no. 260872). M.N.L. received postdoctoral fellowships from EMBO (European Molecular Biology Organization) and FRM (Fondation pour la Recherche Medicale, France).
History
Citation
Science Signaling, 2019, 12 (580), eaav9504Author affiliation
/Organisation/COLLEGE OF LIFE SCIENCES/School of Medicine/Department of Infection, Immunity and InflammationVersion
- AM (Accepted Manuscript)