The Effects of CapZ Peptide (TRTK-12) Binding to S100B–Ca2+ as Examined by NMR and X-ray Crystallography

Structure-based drug design is underway to inhibit the S100B–p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca2+–S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca2+–S100B (1.5-Å resolution) and S100B–Ca2+–TRTK-12 (2.0-Å resolution) determined here indicate that the S100B–Ca2+–TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca2+–S100B involving residues in helices 2 and 3 and loop 2. As with an S100B–Ca2+–p53 peptide complex, TRTK-12 binding to Ca2+–S100B was found to increase the proteinʹs Ca2+-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca2+ ligands and/or improved the Ca2+ coordination geometry of S100B. This possibility was ruled out when the structures of S100B–Ca2+–TRTK-12 and S100B–Ca2+ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD = 0.19). On the other hand, B-factors for residues in EF2 of Ca2+–S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR 15N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca2+–S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca2+–S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.