Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

Michael A Jamros, Leandro C Oliveira, Paul C Whitford, José N Onuchic, Joseph A Adams and Patricia A Jennings

PLOS Computational Biology, 2012, vol. 8, issue 9, 1-8

Abstract: Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell. Author Summary: The Src protein kinases are integral members of numerous signaling pathways involved in cellular growth and differentiation. The master regulator of the Src family is the protein kinase Csk, which adds a phosphate to the C-terminal tail, inhibiting Src Kinase function. Proper regulation of these signaling pathways by Csk is essential as unregulated activity in these pathways is correlated with the development of various cancers and autoimmune diseases. Understanding the nature of the mechanism and structure of Csk may lead to therapeutics and a better understanding of Src signaling pathways. Conformational changes associated with nucleotide binding and release have been shown to regulate the efficiency of Src down-regulation by Csk. To obtain insights into the nature of these nucleotide-induced structural changes, we examined the conformation of Csk in solution while bound to the ATP analog AMP-PNP and product ADP using a combination of small angle x-ray scattering and molecular dynamics. Surprisingly, both nucleotides induce extended conformations of Csk compared to the apo-enzyme, suggesting a novel mode of function. Further understanding of this mode of function may aid in the design of cancer therapeutics that act by regulating Src signaling pathways by modulating the function of Csk.

Date: 2012
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pcbi00:1002695

DOI: 10.1371/journal.pcbi.1002695

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