STRUCTURE/FUNCTION OF S100 PROTEIN

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During the last 3 years we (i) prepared overexpression vectors that produce high yields (greater than 30 mg/liter) of S100B (beta beta), S100A1, and S100L, (ii) determined the solution structures of apo- and Ca2+-bound S100B (beta beta) at high resolution using heteronuclear multidimensional NMR spectroscopy, (iii) collected NMR data in liquid crystalline media necessary to measure dipolar coupling values, (iv) showed that dimeric S100B (beta beta) is the physiologically relevant oligomerization state of this protein, and (v) determined that S100B (beta beta) inhibits p53 phosphorylation by protein kinase C kinase C (PKC) in a Ca2+-dependent manner, and that this inhibition is the result of S100B (beta beta) interacting directly with the C-terminal regulatory domain of p53. We plan to continue characterizing the Ca2+-dependent interaction of S100B (beta beta) with target proteins. First, we will refine our previous NMR structures of apo- and Ca2+-loaded S100B (beta beta) using dipolar coupling constraints. We will also determine the 3D structure of Ca2+-bound S100B (beta beta) complexed with peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). This will represent the first 3D structure of a S100-target protein complex. The binding of Zn2+ to S100B (beta beta) will also be characterized, and we will determine whether the Zn2+ binding site overlaps with the target protein site. Heteronuclear relaxation measurements are planned for all of the structures that we solve in order to clarify how Ca2+ and target protein binding affects dynamic processes in S100B (beta eta). Lastly, the 3D solution structures of S100A and S100L will be determined and compared to S100B (beta beta). This will be done with the goal of identifying the structural properties of these two proteins that lead to their higher affinity for Ca2+ and their specificity in target protein binding. Our effort is directed towards characterizing S100-target protein interactions with the long-range goal of inhibiting them. Therefore, structural studies, together with site-directed mutagenesis, thermodynamic binding, and dynamic measurements will be used to characterize, at atomic resolution, the interaction between specific residues of S100B (beta beta) with those of various protein targets in solution. An inhibitor based on this information could be relevant to treating uncontrolled cell growth found in diseases such as cancer and Alzheimer's disease.