title: Substrate-specific reorganization of the conformational ensemble of CSK implicates novel modes of kinase function.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Jamros
authors.forename: Leandro C
authors.initials: LC
authors.lastname: Oliveira
authors.forename: Paul C
authors.initials: PC
authors.lastname: Whitford
authors.forename: José N
authors.initials: JN
authors.lastname: Onuchic
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Jennings
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000081247:CSK Tyrosine-Protein Kinase; D003198:Computer Simulation; D004789:Enzyme Activation; D008956:Models, Chemical; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D000255:Adenosine Triphosphate; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases