Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα

Sangbae Lee, Titu Devamani, Hyun Deok Song, Manbir Sandhu, Adrien Larsen, Ruth Sommese, Abhinandan Jain, Nagarajan Vaidehi, Sivaraj Sivaramakrishnan

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Protein kinase Cα (PKC) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKC, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein–protein interaction has limited structural mapping of kinase–substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase–substrate interface. We found that the binding strength of the kinase–substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate– kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKC for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase–substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases.

Original languageEnglish (US)
Pages (from-to)16300-16309
Number of pages10
JournalJournal of Biological Chemistry
Volume292
Issue number39
DOIs
StatePublished - Sep 29 2017

Bibliographical note

Funding Information:
This work was supported in part by National Institutes of Health Director’s New Innovator Award 1DP2 CA186752-01 (to S. S.) and the Beckman Research Institute of the City of Hope (to N. V.). The authors declare that they have no conflicts of interest with the contents of this article. The con-tent is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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