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

Edited by Natalia Jura, University of California, San Francisco, CA, and accepted by Editorial Board Member Brenda A. Schulman November 10, 2017 (received for review June 27, 2017)
December 5, 2017
114 (51) 13453-13458

Significance

Phosphorylation is a ubiquitous modification that has been implicated in signaling and other functions, but the atomic-level mechanisms are not completely understood. We identify a salt-bridge competition or “theft” mechanism wherein a phosphoserine, but not a phosphomimetic, breaks a pre-existing 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. These findings identify a facile and evolutionarily accessible mechanism for reorganizing salt bridges and other electrostatic networks with only a single mutation to trigger a functional switch.

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.

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Data Availability

Data deposition: The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank, www.rcsb.org [PDB ID codes 6ENS (RKIP) and 6ENT (Δ143-146 variant)].

Acknowledgments

We thank Helmholtz-Zentrum Berlin for the allocation of synchrotron radiation beamtime and the staff of beamline MX 14.1 for technical assistance and Drs. Gianluigi Veglia and Jonggul Kim for valuable discussions. This work was supported by Grants GM087630 (to M.R.R.), GM55694 (to T.R.S.), Deutsche Forschungsgemeinschaft FZ82 (to K.L., C.K., and H.S.) and SFB688 and TPA17 (to K.L.), the German Ministry of Research and Education and the Ministry for Innovation, Science and Research of the Federal State of North Rhine-Westphalia (K.L.).

Supporting Information

Appendix (PDF)

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 114 | No. 51
December 19, 2017
PubMed: 29208709

Classifications

Data Availability

Data deposition: The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank, www.rcsb.org [PDB ID codes 6ENS (RKIP) and 6ENT (Δ143-146 variant)].

Submission history

Published online: December 5, 2017
Published in issue: December 19, 2017

Keywords

  1. phospho-swap
  2. protein interaction
  3. salt-bridge competition
  4. Raf Kinase Inhibitory Protein
  5. conformational change

Acknowledgments

We thank Helmholtz-Zentrum Berlin for the allocation of synchrotron radiation beamtime and the staff of beamline MX 14.1 for technical assistance and Drs. Gianluigi Veglia and Jonggul Kim for valuable discussions. This work was supported by Grants GM087630 (to M.R.R.), GM55694 (to T.R.S.), Deutsche Forschungsgemeinschaft FZ82 (to K.L., C.K., and H.S.) and SFB688 and TPA17 (to K.L.), the German Ministry of Research and Education and the Ministry for Innovation, Science and Research of the Federal State of North Rhine-Westphalia (K.L.).

Notes

This article is a PNAS Direct Submission. N.J. is a guest editor invited by the Editorial Board.

Authors

Affiliations

John J. Skinner1
Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637;
Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637;
Present address: iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
Sheng Wang1
Toyota Technological Institute at Chicago, Chicago, IL 60637;
Department of Genetics, University of Chicago, Chicago, IL 60637;
Present address: Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia.
Jiyoung Lee
Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637;
Colin Ong
Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637;
Ruth Sommese
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455;
Sivaraj Sivaramakrishnan https://orcid.org/0000-0002-9541-6994
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455;
Wolfgang Koelmel
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany;
Maria Hirschbeck
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany;
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany;
Caroline Kisker
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany;
Kristina Lorenz
Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany;
Cardiovascular Pharmacology, Leibniz-Institut für Analytische Wissenschaften (ISAS), 44139 Dortmund, Germany;
West German Heart and Vascular Center Essen, University Hospital Essen, 45122 Essen, Germany
Tobin R. Sosnick4 [email protected]
Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637;
Marsha Rich Rosner4 [email protected]
Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637;

Notes

4
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: J.J.S., K.L., T.R.S., and M.R.R. designed research; J.J.S., S.W., J.L., C.O., W.K., M.H., H.S., and C.K. performed research and analyzed data; R.S., S.S., and K.L. contributed new reagents/analytic tools; J.J.S., S.W., T.R.S., and M.R.R. analyzed data; and J.J.S., T.R.S., and M.R.R. wrote the paper.
1
J.J.S. and S.W. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

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    Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome
    Proceedings of the National Academy of Sciences
    • Vol. 114
    • No. 51
    • pp. 13303-E11060

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