Research ArticleStructural Biology

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

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Science Signaling  07 May 2019:
Vol. 12, Issue 580, eaav9504
DOI: 10.1126/scisignal.aav9504

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An FHA domain with dual specificity

Forkhead-associated (FHA) domains participate in phosphorylation-dependent signaling pathways by binding to phosphothreonine. As part of a signaling pathway that controls glutamate metabolism, the mycobacterial FHA domain–containing protein GarA binds to both phosphorylated upstream partners, such as the kinases PknB and PknG, and nonphosphorylated downstream partners, such as the 2-oxoglutarate decarboxylase KGD. Through biochemical and structural studies, Wagner et al. found that the interactions of GarA with both phosphorylated PknB and nonphosphorylated KGD were mediated by the phosphate-binding pocket of the FHA domain, which bound to a phosphothreonine in the activation loop of PknB and to a phosphomimetic aspartate residue in KGD. In addition to illustrating the dual binding specificity of GarA, these findings demonstrate a physiological role for aspartate as a phosphomimetic.

Abstract

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.

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