Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk.
The SH2 domain is required for high catalytic activity in the COOH-terminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the K(m) for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated K(m) values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen-deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
Lilly
L
Wong
Scot
S
Lieser
Barbara
B
Chie-Leon
Osamu
O
Miyashita
Brandon
B
Aubol
Jennifer
J
Shaffer
Josè N
JN
Onuchic
Patricia A
PA
Jennings
Virgil L
VL
Woods
Joseph A
JA
Adams
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020134:Catalytic Domain; D019295:Computational Biology; D003903:Deuterium / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D009711:Nucleotides; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D003903:Deuterium; D019061:src-Family Kinases