Experimental and Calculated Shift in pKa upon Binding of Phosphotyrosine Peptide to the SH2 Domain of p56lck

Abstract

The pH dependence of the affinity of a 11-mer phosphotyrosine (pY) peptide (EPQpYEEIPIYL-NH2) for the SH2 domain of the tyrosine kinase p56lck was investigated with surface plasmon resonance (SPR). From SPR competition experiments the affinity in solution was obtained. The pH dependence of the affinity in solution can be well described by a proton linkage model with a single pKa shift upon binding, from 6.1 to 4.7. This shift is ascribed to the transition from the −2 to the −1 ionisation state of the tyrosine phosphate group. Based on the X-ray structure for the complex with Lck SH2, a pKa value of 5.3 for the bound pY peptide was computed, modelling the solvated protein as a system of point charges in a continuum. With the phosphate in the −2 state the binding energy is 1.8 kcal/mol more favourable than for the −1 state, corresponding to a 20-fold higher affinity. A proper charge is relevant in the design of potential therapeutic Lck SH2 ligands with mimics for the metabolically unstable tyrosine phosphate group.