Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS).
Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.
Hamuro
Yoshitomo
Y
Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla 92093-0656, USA.
Wong
Lilly
L
Shaffer
Jennifer
J
Kim
Jack S
JS
Stranz
David D
DD
Jennings
Patricia A
PA
Woods
Virgil L
VL
Adams
Joseph A
JA
D000227:Adenine Nucleotides / Q000378:metabolism; D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D020134:Catalytic Domain; D003903:Deuterium; D006859:Hydrogen; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases
D000227:Adenine Nucleotides; D010446:Peptide Fragments; D006859:Hydrogen; D003903:Deuterium; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases