Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

Abstract

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

The labile interactions of cyclic electron flow effector proteins

“…(ii) An alternative to cross-link-mediated alterations in PETO across the membrane would be that of an STT7-induced phosphorylation of stromal Ser/Thr in both PETO and su-IV, which have been identified in proximity to our reported cross-link site (28). These posttranslational modifications could reorganize salt bridges and other electrostatic networks (51), which would then favor stromal PETO-Cyt-f interactions as well. Here, Fig.…”

Targeting Raf Kinase Inhibitory Protein Regulation and Function

“…Most notably, phosphorylation of Ser153 residue by protein kinase C (PKC)switches RKIP’s binding from Raf to G-protein-coupled receptor kinase 2 (GRK2) [ 14 ], resulting in activation of protein kinase A (PKA). The mechanism by which this phospho-switch occurs involves a novel salt bridge theft that enables the promotion or disruption of peptide interactions to provide specificity at protein interfaces [ 15 , 16 ]. This illustrates RKIP’s potential role in sensing active signaling networks and modifying the cellular kinome through new protein interactions.…”

Triple Arginines as Molecular Determinants for Pentameric Assembly of the Intracellular Domain of 5-HT3A Receptors

“…We do not know exactly why the ΔMA construct forms trimers under the given experimental conditions nor do we know the exact driving forces behind dimerization of the MA and QDA constructs in solution. It is a possibility, as documented elsewhere (65), that the breakage of pre-existing salt-bridges can induce protein remodeling via initiation of partial unfolding and reorganization of electrostatic interactions leading to the formation of non-native oligomers. Nevertheless, it is important to emphasize that we did not observe pentamers formed by any of the engineered proteins during our investigation, with the notable exception of ICD and Δ44.…”

“…In this regard, it is intriguing to find that the 5-HT 3A -ICD is structured as pentamers in the absence of both the ECD and the TMD (44). Multiple studies underscore the importance of salt-bridge formation in the stabilization of a protein oligomeric assembly (63-65). Therefore, we interrogated whether some of the aforementioned function-governing salt bridges additionally play a fundamental role in pentameric assembly.…”