Contribution of an active site cation-pi interaction to the spectroscopic properties and catalytic function of protein tyrosine kinase Csk.
Csk is a soluble protein tyrosine kinase that phosphorylates and negatively regulates protein tyrosine kinases of the Src family. The spectral properties of the intrinsic Trp fluorescence of Csk and their underlying structural features were investigated in combination with urea denaturation and site-specific mutagenesis. It was found that W352 contributed approximately 35% of the total Trp fluorescence of Csk even though seven other Trp residues were present. The enhanced contribution by W352 to Csk fluorescence was due to an interaction between its indole ring and the positively charged guanidino group of R318. W352 is located on the peptide substrate binding P+1 loop, and R318 is located on the catalytic loop. The W352-R318 interaction, called a cation-pi interaction, uniquely couples the two loops in the active site. Mutations that disrupted this coupling resulted in varying levels of decreases in Csk activity, and consistent and significant increases in K(m) values for its physiological substrate, Src protein tyrosine kinase. These results indicated that structural coupling between the two loops by the cation-pi interaction played an important role in Csk substrate binding. Since both R318 and W352 are highly conserved among protein tyrosine kinases, this cation-pi interaction is likely a signature structural feature of most, if not all, PTKs. These studies elucidated the roles of two conserved signature residues in Csk and formed a baseline for further structure-function studies of Csk and other PTKs.
D000818:Animals; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D002384:Catalysis; D002645:Chickens; D011505:Protein-Tyrosine Kinases; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D019061:src-Family Kinases / Q000737:chemistry*
D014364:Tryptophan; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases