DETERMINATION OF AFFINITIES FOR LCK SH2 BINDING PEPTIDES USING A SENSITIVE FLUORESCENCE ASSAY - COMPARISON BETWEEN THE PYEEIP AND PYQPQP CONSENSUS SEQUENCES REVEALS CONTEXT-DEPENDENT BINDING-SPECIFICITY

The development of a sensitive fluorescence binding assay for evaluati ng the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides c ontaining the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were ch aracterized in the presence and absence of the lck SH2 domain. The bin ding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(A ANS)QpYEEIP exhibited the highest binding affinity (K-d 3.98 x 10(-8) M) and largest change in emission intensity (approximate to 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in compe titive binding assays to study the interaction of the lck SH2 domain w ith a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY(505) C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discusse d in terms of binding affinities. Comparison between the two peptide s eries revealed that the contributions of individual substitutions to b inding affinity are context-dependent. The data also led to the conclu sion that the presence of P at +2 results in a functional ''truncation '' of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding, This implicit truncation may actually be a desired property for the autoregulatory nature of t he pYQPQP sequence, since it retains specificity for the SH2, domain w hile adjusting the Kd to a value appropriate for maintaining the delic ate balance of receptor-ligand interactions that are involved in signa l transduction events.