Joshua Ward - Constraining Binding Hot Spots: NMR and Molecular Dynamics Simulations Provide a Structural Explanation for Enthalpy−Entropy Compensation in SH2−Ligand Binding

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    Joshua M. Ward,   Nina M. Gorenstein,   Jianhua Tian,   Stephen F. Martin   and Carol Beth Post;   J. Am. Chem. Soc.,    2010,    132   (32), pp 11058–11070


    NMR spectroscopy and molecular dynamics (MD) simulations were   used to probe the structure and dynamics of complexes of three   phosphotyrosine-derived peptides with the Src SH2 domain in an   effort to uncover a structural explanation for enthalpy−entropy   compensation observed in the binding thermodynamics. The series   of phosphotyrosine peptide derivatives comprises the natural   pYEEI Src SH2 ligand, a constrained mimic, in which the   phosphotyrosine (pY) residue is preorganized in the bound   conformation for the purpose of gaining an entropic advantage to   binding, and a flexible analogue of the constrained mimic. The   expected gain in binding entropy of the constrained mimic was   realized; however, a balancing loss in binding enthalpy was also   observed that could not be rationalized from the crystallographic   structures. We examined protein dynamics to evaluate whether the   observed enthalpic penalty might be the result of effects arising   from altered motions in the complex.15N-relaxation   studies and positional fluctuations from molecular dynamics   indicate that the main-chain dynamics of the protein show little   variation among the three complexes. Root mean squared (rms)   coordinate deviations vary by less than 1.5 Å for all   non-hydrogen atoms for the crystal structures and in the ensemble   average structures calculated from the simulations. In contrast   to this striking similarity in the structures and dynamics, there   are a number of large chemical shift differences from residues   across the binding interface, but particularly from key Src SH2   residues that interact with pY, the “hot spot” residue, which   contributes about one-half of the binding free energy. Rank-order   correlations between chemical shifts and ligand binding enthalpy   for several pY-binding residues, coupled with available   mutagenesis and calorimetric data, suggest that subtle structural   perturbations (<1 Å) from the conformational constraint of the   pY residue sufficiently alter the geometry of enthalpically   critical interactions in the binding pocket to cause the loss of   binding enthalpy, leading to the observed enthalpy−entropy   compensation. We find no evidence to support the premise that   enthalpy−entropy compensation is an inherent property and   conclude that preorganization of Src SH2 ligand residues involved   in binding hot spots may eventuate in suboptimal interactions   with the domain. We propose that introducing constraints   elsewhere in the ligand could minimize enthalpy−entropy   compensation effects. The results illustrate the utility of the   NMR chemical shift to highlight small, but energetically   significant, perturbations in structure that might otherwise go   unnoticed in an apparently rigid protein.

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