The C-terminus and linker region of S100B exert dual control on protein-protein interactions with TRTK-12

S100B, an EF-hand calcium-binding protein composed of two S100β monomers, undergoes a calcium-dependent conformational change that provides a surface for target interactions. In this study, the calcium-sensitive S100B-binding epitope TRTK-12 has been used to probe the contributions of the linker and C-terminal regions of S 100B to protein-protein interactions. These contributions were quantified using C-terminal mutant S100B proteins lacking the C-terminal seven (S100B85stop) or nine (S100B83stop) residues or containing alanine substitutions at Phe87 (F87A), Phe88 (F88A), or both (F8788A). Both F8788A and F88A bound TRTK-12 less tightly (K_d = 1.85 ± 0.02 and 0.97 ± 0.08 μM, respectively) than the wild-type protein (K_d = 0.27 ± 0.03 μM, ΔG = -37.2 kJ/mol), indicating these residues are important for TRTK-12 interaction. The truncated S100B proteins bound TRTK-12 much more weakly (K_d = 659.7 ± 119.3 ΜM, ΔG = -17.9 kJ/mol), indicating the linker region contributed about 50% to the binding of TRTK-12, while the C-terminus contributed the remaining 50% of the binding energy. Based on mutagenesis and NMR chemical shift studies, a comparison with known S100-target protein complexes showed the S100B-TRTK-12 complex has the strongest resemblance to the S100A10-annexin II interaction.