Protein Dynamics of S100B and S100B D63N in the Presence and Absence of a CapZ-derived Peptide (TRTK-12)

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. In malignant melanomas, S100B contributes to cell proliferation by binding to p53 and inhibiting its tumor suppressing properties. One property of S100B is that it only binds to calcium tightly when its target protein is present, a feature that drug design studies for restoring p53 in melanoma must mimic.   The purpose of this study is to determine the mechanism for how target protein binding to S100B causes an increase in calcium-binding affinity.  The mechanism behind this 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 EF-hand of S100B for NMR side-chain dynamics (+/-TRTK-12).  We found evidence of protein dynamics in side-chain and backbone amides using ¹⁵N-backbone dynamic (T1, T2, and hetNOE) and relaxation dispersion experiments.  In the backbone, motion was detected in the hinge region and C-terminus of helix-4 at multiple timescales in the absence of TRTK-12.  When TRTK-12 was introduced, the majority of the dynamics seen in backbone and side chain amides were drastically reduced.  This suggests that in the presence of TRTK-12, S100B undergoes a decrease in protein dynamics that translates from the site of target binding to the EF-hand.