| NMR spectroscopy and molecular dynamics simulations were used to examine the binding of a canonical phosphorylated tyrosine (pY) peptide substrate (pYEEI), a conformationally constrained pY analog (DeltapYEEI), and a flexible pY analog (fpY-EEI) to the pp60 v-Src SH2 domain (Src SH2). The binding series has been previously shown by isothermal titration calorimetry (ITC) measurements to exhibit enthalpyentropy compensation, such that the desired favorable change in the free energy of binding (DeltaG°) is not observed despite the achievement of the expected gain in binding entropy (Delta S°). A structural explanation for the enthalpic compensation could not be rationalized, as no significant differences were observed between crystal structures of the SH2-pYEEI and SH2--DeltapYEEI complexes. Chemical shift perturbation analysis reveals a number of strong, constraint-dependent differences between the three complexes. These differences are scattered across the binding interface and central beta-strand, revealing that the constraint induces long-distance effects in the dynamic structure of the protein in solution. The SH2 main-chain dynamics in each complex were characterized by 15N relaxation, which revealed notable similarities between the three bound states. Supplementary to the experimental data, molecular dynamics (MD) also indicate similar isotropic atomic fluctuations, simulated NMR order parameters, and vibrational modes. Analysis of anisotropic fluctuations from the simulations suggests that while the aforementioned parameters indicate a common dynamic amplitude for atoms the three complexes, the vibration of the atoms can possess appreciable directionality that differs in the presence of each bound peptide. Based on these results, the unfavorable change in enthalpy is proposed to be an accumulation of small perturbations associated with sub-1 A differences in geometry and principle fluctuation tensor within the pY-binding pocket and central beta-sheet that are unobservable at the resolution of the X-ray structures. |
