Role of Cys residues of C-terminal SH2 domain of phosphoinositide 3-kinase in its conformational stability and CD28-binding ability

Abstract

Proteins fold into their most stable yet dynamic structures that enable their functions and exist in a balance between stability and instability. Protein folding and binding can be evaluated based on their thermodynamics determined using various methods. In this study, we analyzed the folding and binding thermodynamics of the C-terminal Src homology 2 domain (cSH2) of phosphoinositide 3-kinase and its Cys mutants, C656S, C659V, C670L, and C656S/C659V/C670L. The protein binds to the consensus sequence including phosphorylated Tyr, and the binding affinity to CD28-derived phosphopeptides decreased upon the Cys mutation: wild-type > C656S > C659V > C670L > C656S/C659V/C670L. The decreased affinity was mainly due to the increased binding enthalpy change. The thermal stability analysis showed the opposite tendency: wild-type < C656S < C659V < C670L < C656S/C659V/C670L, indicating a trade-off in protein architecture between stability and binding function. Based on the crystal structures of the cSH2 wild-type and C656S/C659V/C670L, the increased stability could be explained by the increased hydrophobic contacts. The wild-type protein would have evolved to have a native structure while maintaining the dynamics to express its binding function. Furthermore, a unique unfolding transition was observed for the cSH2 wild-type, possibly due to Cys oxidation, which was significantly changed upon the single mutation of Cys residues. We also demonstrated the validity of various methods for analyzing protein stability.