Abstract 2328: 15N-backbone and side-chain dynamics of the mutant calcium-binding protein S100B D63N
Melissa A. Liriano, Kristen M. Varney, Rieko Ishima, David J. Weber · Cancer Research · 2011
Abstract Similar to calmodulin and other EF-hand-containing proteins, S100 proteins have no inherent enzymatic activity and perform their biological functions via calcium-dependent protein-protein interactions. Elevated protein levels of S100B have been linked to various types of cancers, including malignant melanomas, glioblastomas, and anaplastic astrocytomas. In the case of malignant melanomas, S100B contributes to cell proliferation by binding to p53 and inhibiting its tumor suppressing properties. However, one property of S100 proteins, including S100B, is that it only binds to calcium tightly when its target protein is bound, a feature that drug design studies for restoring p53 in melanoma must mimic. Therefore, the purpose of this study is to determine the mechanism for how target protein binding to S100B causes an increase in calcium-binding affinity. Previous studies suggest that the increase in calcium affinity in the presence of target is not the result of a structural modification. As an alternative explanation, the mechanism behind this event may involve a reduction in the ligand dynamics coordinating calcium in the canonical EF-hand when target peptide is present. To test this hypothesis with S100B, an asparagine mutant was made in order to have an 15N-probe in the tight-binding EF-hand of S100B for NMR side-chain dynamics with and without target peptide. 15N backbone dynamic experiments (T1, T2, and hetNOE) were collected for the S100B D63N mutant (+/- TRTK-12) at two magnetic fields to determine the effects of peptide binding at multiple time scales. Relaxation dispersion experiments were also collected for backbone and side-chain amides of S100B in the presence and absence of TRTK-12. For the backbone dynamic experiments, motion was detected in the hinge region and C-terminus of helix-4 at multiple timescales in the absence of TRTK-12 (ns-sec timescale). A similar profile was observed for those backbone residues undergoing motion in the chemical exchange time scale seen with the relaxation dispersion data. However, when a molecular target was introduced, the majority of these backbone amides that make up the hydrophobic pocket displayed a general decrease in dynamics. Side chain dynamics in the EF-hand was appreciated in the absence of TRTK-12 peptide, however when the target was present, no chemical exchange was seen with the 15N-D63N probe in the EF-hand. These findings imply that In the presence of target peptide, S100 proteins undergo a decrease in protein dynamics that translates from the site of target binding to the EF-hand. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2011-2328