S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.

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 Ca 2+ -S100B and a target, TRTK-12, which binds to the p53 binding site on S100B. The structures of Ca 2+ -S100B (1.5 Å resolution) and S100B-Ca 2+ -TRTK12 (2.0 Å resolution) determined here indicate that the S100B-Ca 2+ -TRTK12 complex is dominated by an interaction between Trp-7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca 2+ -S100B involving residues in helices 2 & 3 and loop 2. As with a S100B-Ca 2+ -p53 peptide complex, TRTK-12 binding to Ca 2+ -S100B was found to increase the proteins Ca 2+ ion binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca 2+ ligands and/or improved Ca 2+ ion coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca 2+ -TRTK12 and S100B-Ca 2+ 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 Ca 2+ -S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR 15 N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca 2+ -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 Ca 2+ -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.

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca 2+ -loaded and Zn 2+ ,Ca 2+ -loaded S100B were determined by X-ray crystallography at 2.15 Å (R free = 0.266) and 1.85 Å (R free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca 2+ -loaded S100B and Zn 2+ ,Ca 2+ -loaded S100B determined here (1.88 Å; R free = 0.267). In the presence and absence of Zn 2+ , electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (±Zn 2+ ), and the second Pnt molecule was mapped to the dimer interface (site 2; ±Zn 2+ ) and in a pocket near residues that define the Zn 2+ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn 2+ to Ca 2+ -S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn 2+ ,Ca 2+ -S100B when compared to Pnt-Ca 2+ -S100B. That Pnt can adapt to this Zn 2+ -dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca 2+ -and Ca 2+ ,Zn 2+ -bound S100B.

Mutations in the second EF-hand (D61N, D63N, D65N, E72A) of S100B were used to study its Ca2+-binding and dynamic properties in the absence and presence of abound 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; 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 were increased by 5- to 19-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; 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 nuclear magnetic resonance (NMR). Likewise, backbone motions on multiple time scales (ps-ms) 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, so 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. Thus, a model for how S100B-TRTK12 complex formation increases Ca2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.

S100A4, a member of the S100 family of Ca 2þ -binding proteins, regulates carcinoma cell motility via interactions with myosin-IIA. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Using a unique biosensorbased assay, trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction. To examine the interaction of S100A4 with TFP, we determined the 2.3 Å crystal structure of human Ca 2þ -S100A4 bound to TFP. Two TFP molecules bind within the hydrophobic target binding pocket of Ca 2þ -S100A4 with no significant conformational changes observed in the protein upon complex formation. NMR chemical shift perturbations are consistent with the crystal structure and demonstrate that TFP binds to the target binding cleft of S100A4 in solution. Remarkably, TFP binding results in the assembly of five Ca 2þ -S100A4/TFP dimers into a tightly packed pentameric ring. Within each pentamer most of the contacts between S100A4 dimers occurs through the TFP moieties. The Ca 2þ -S100A4/prochlorperazine (PCP) complex exhibits a similar pentameric assembly. Equilibrium sedimentation and cross-linking studies demonstrate the cooperative formation of a similarly sized S100A4/TFP oligomer in solution. Assays examining the ability of TFP to block S100A4-mediated disassembly of myosin-IIA filaments demonstrate that significant inhibition of S100A4 function occurs only at TFP concentrations that promote S100A4 oligomerization. Together these studies support a unique mode of inhibition in which phenothiazines disrupt the S100A4/ myosin-IIA interaction by sequestering S100A4 via small molecule-induced oligomerization.calcium | X-ray crystallography | NMR | small molecule inhibitor | metastasis T he S100 proteins, of which there are more than 20 members, are characterized by their solubility in 100% saturated ammonium sulfate (1 and 2). Each S100 family member contains two Ca 2þ -binding loops; a C-terminal "typical" EF-hand comprised of 12 residues and an N-terminal pseudo EF-hand consisting of 14 residues. The basic organization of the S100 proteins is a symmetric, antiparallel homodimer, in which the N-and C-terminal helices (helices 1 and 4) from each subunit interact to form a stable four helix bundle that serves as the dimer interface. Calcium binding to the C-terminal typical EF-hand significantly alters the angle between helices 3 and 4, which flank the C-terminal Ca 2þ -binding loop, and exposes a hydrophobic cleft that constitutes a binding surface for target proteins (3-5). Thus the S100 proteins operate as calcium-activated switches that bind and regulate the activity of diverse protein targets. S100 proteins are expressed in a tissue and cell specific manner. Elevated expression of individual family members is associated with a number of human pathologies, including cardiomyopathies, cancer, neurodegeneration, and in...