Study of Sequence Dynamics Relationship with Binding Affinity of Pin1-WW Domain

A molecular understanding of protein-protein interactions is important to be able to understand cell cycle and diseases associated with it. The specificity of these protein- protein interactions is usually associated with the chemical properties of the amino acids that contact the substrate. This thesis investigates the dependence of protein binding affinity on the sequence specific dynamics of the protein binding loop. The model system used for this study is WW domain of Pin1. Pin1 protein is a two domain protein that interacts with other proteins important for cell cycle. It catalyzes cis-trans isomerization of p-Ser/Pro or p-Thr/Pro motifs. Generally, WW domains are involved in the binding of polyproline II helix motifs to the surface of the protein whose binding preference is found to depend on recognition specificity loop. In the case of Pin1-WW, this loop prefers the p-Ser/Pro or p-Thr/Pro motifs. To establish the relationship between the dynamics of the sequence and the binding affinity, ps-ns motion study using NMR spectroscopic method has been done on the apo wild-type WW and its mutants. The mutants were chosen by deleting or substituting one or more residues in the loop I region of the WW domain. The key residues responsible for binding specificity are ser16-arg17-ser18-ser19- gly20-arg21, and so the mutants involved changes in these residues keeping arg17 intact whose side chain has been shown to be involved in forming direct contacts with substrate. The plan is to study whether the loop region dynamics changes even though we do not alter the residue or the side chains directly involved in bonding and thus showing that chemical nature of the sequence alone does not govern the binding preference of the domain. The binding affinity for the wild-type [1] and WW mutants has been calculated (by Tao Peng) and the Kd value for wild-type (wt) is observed to be much less than its mutants for the substrate cdc25. The expected result from the dynamic study would be to see a decrease in the motion of the loop region for the mutants of WW and a similar dynamic pattern for its orthologs on the basis of the hypothesis that nature possibly selects for conformational dynamics as well. To investigate and make a quantitative assessment of the dynamic-binding relationship, the dynamic information for each of the sites in the protein will be extracted from the relaxation rates R1 (longitudinal rate constant), R2 (transverse rate constant), R1̏ (rotating frame spin lattice rate constant) and steady state heteronuclear NOE, measured using NMR at 700 MHz and 800 MHz field strengths.The rate constants will be analyzed using the Lipari-Szabo model [2]; the analysis will provide the site-specific dynamic parameters that describe the internal dynamics of the Pin1-WW domain. These motional parameters will be determined by non-linear least squares fitting method of the relaxation rates to model functions. The fitted parameters will contain the information on the amplitude and the time-scale of the internal motion of the backbone N-H bonds. A comparison of the dynamic parameters will be done for the wt-WW and its mutants and then correlated with their binding affinity .