During the last 3 years, (i) high yields (>30 mg purified protein/liter media) of S 100B, S 100A 1, S 100A3, CAN19, mtsl, p53, p53(324-393), p53(303-393), p53(303-367), hepatitis B viral protein X (HBVX), SUMO-1, C-terminal fragment of myosin IIA(1900-1961), and the HMG A box of the high mobility group protein- 1 (HMG-1) were prepared in minimal media as necessary for isotopic labeling (2H, 13C, 15N, etc). (ii) We determined the solution structures of apo-S 100B, apo-S 100A 1, calcium-bound S 100B, and calcium-bound S 100B in a complex with the negative regulatory domain of p53 (residues 367-388). (iii) We showed that dimeric S 100B is the physiologically relevant oligomerization state of S 100B, and that S 100B inhibits protein kinase C (PKC) phosphorylation of p53. (iv) We developed cellular-based assays for p53 and showed that p53 function is inhibited in tumor cell lines as a result of the calcium-dependent p53-S 100B interaction. This interaction includes both the oligomerization and negative regulatory domains at the C-terminus of p53. We plan to continue characterizing the calcium-dependent interaction of S 100B with the tumor suppressor protein p53. The effects that p53 phosphorylation, acetylation, and sumoylation have on S 100B binding will be examined. The binding of zinc to S 100B and heterodimer formation (i.e. S 100A 1/S 100B, CaN 19/S 100B and mts 1/S 100B etc.) will also be characterized. These data are necessary to determine whether covalent modifications of p53 and/or other S100B binding events affect S100B-p53 complex formation and function We will also determine the 3D structure of calcium-bound S100B complexed with a larger construct of p53 that includes both the oligomerization and the C-terminal regulatory domains of p53 (residues 324-393; Kd=24nM). Heteronuclear relaxation measurements for backbone and sidechain resonances are planned for all of the structures that we solve (or have solved) in order to clarify how calcium and p53 binding affects the dynamics of S100B (and p53). These dynamic data will be used in a search for small molecule inhibitors of the S 100B-p53 interaction. Lastly, the 3D solution structures of other proteins that bind the C-terminus of p53 will be examined including the metastasis protein 1 (mts 1), S100A3, the CaN 19 tumor suppressor, the hepatitis B viral protein (HBVX), the p53 binding domain of BLM, and the A box ofHMG-1. It will be interesting to determine whether these or other p53-binding proteins enhance and/or compete with the S 100B-p53 interaction.
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