Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 μM), Ca2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 μM, D65NKD = 968 ± 171 μM, and E72AKD = 471 ± 133 μM), when compared to wild-type protein (WTKD = 56 ± 9 μM). For the TRTK-12 complexes, the Ca2 +-binding affinity to wild type (WT + TRTKKD = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (D61N + TRTKKD = 29 ± 1.2 μM, D63N + TRTKKD = 10 ± 2.2 μM, D65N + TRTKKD = 73 ± 4.4 μM, and E72A + TRTKKD = 18 ± 3.7 μM). In addition, Rex, as measured using relaxation dispersion for side‐chain 15N resonances of Asn63 (D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, D61NS100B, D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.