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

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journal contribution
posted on 13.05.2019, 13:35 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 Alzari
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.

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), eaav9504

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/School of Medicine/Department of Infection, Immunity and Inflammation

Version

AM (Accepted Manuscript)

Published in

Science Signaling

Publisher

American Association for the Advancement of Science

eissn

1937-9145

Copyright date

2019

Available date

13/05/2019

Publisher version

https://stke.sciencemag.org/content/12/580/eaav9504

Notes

The atomic coordinates and structure factors of the four crystal structures were deposited in the PDB with codes 6I2P (MtPknBCD,L33A-MtGarA), 6I2Q (MsKGDΔ360-MsGarAΔ44), 6I2R (MsKGDΔ360,R802A-MsGarAΔ44), and 6I2S (MsKGDΔ360,R802A-MsGarAΔ44, 2-oxoglutarate soak). All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. SUPPLEMENTARY MATERIALS stke.sciencemag.org/cgi/content/full/12/580/eaav9504/DC1 Fig. S1. Alignment of GarA homologs in selected Actinobacteria. Fig. S2. Continuous sedimentation coefficient distribution analysis of the PknBCD-GarA complex. Fig. S3. ITC characterization of the interaction between autophosphorylated PknBCD and GarA. Fig. S4. The N-terminal GarA extension occupies a similar position in different GarA structures. Fig. S5. Conserved mode of phosphopeptide recognition in different M. tuberculosis FHA domains. Fig. S6. Detailed structure of the PknB activation loop bound to GarA. Fig. S7. Formation of the enamine-ThDP covalent adduct in the presence of GarA. Fig. S8. SPR studies of protein-protein interactions. Table S1. Data collection and refinement statistics. References (64, 65)

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en

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