The effects of CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography

Abstract

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 TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca(2+)-S100B (1.5-A resolution) and S100B-Ca(2)(+)-TRTK-12 (2.0-A resolution) determined here indicate that the S100B-Ca(2+)-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca(2+)-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca(2)(+)-p53 peptide complex, TRTK-12 binding to Ca(2+)-S100B was found to increase the protein's Ca(2)(+)-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 the Ca(2+) coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca(2+)-TRTK-12 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.

Figure 1

Thermodynamic binding constants for S100B-metal and S100B-metal-TRTK12 complexes. (a) Displacement of the TAMRA-TRTK12 peptide from the S100B-Ca²⁺-TAMRA-TRTK12 complex by unlabeled TRTK12 as monitored by fluorescence polarization. The solution contained 1.5 μM S100B, 50 nM TAMRA-TRTK12, and 10 mM CaCl₂ in 50mM Tris-HCl pH 7.5. (a, inset) Binding of TAMRA-TRTK12 to S100B-Ca²⁺ as monitored by fluorescence polarization. The solution contained 50 nM TAMRA-TRTK12, and 10 mM CaCl₂ in 50 mM Tris-HCl, pH 7.5. (b) Displacement of the TAMRA-TRTK12 peptide from the S100B-Mn²⁺-TAMRA-TRTK12 complex by unlabeled TRTK12 as monitored by changes in fluorescence polarization (ΔmP). The solution contained 4 μM S100B, 50 nM TAMRA-TRTK12, and 10 mM MnCl₂ in 50mM Tris-HCl pH 7.5. (b, inset) Binding of TAMRA-TRTK12 to S100B-Mn²⁺ as monitored by changes in fluorescence polarization. The solution contained 50 nM TAMRA-TRTK12, and 10 mM MnCl₂ in 50 mM Tris-HCl pH 7.5. (c) Displacement of bound Mn²⁺ by Ca²⁺ from S100B in the presence of TRTK as detected by EPR and NMR (inset). The solutions for both experiments contain 65 μM S100B, 150 μM TRTK-12, and 82 μM MnCl₂ in 50mM Tris-HCl pH 7.5. The data were fit with the Hill equation to determine K_app with the corresponding dissociation constants calculated using competition equations described in Material and Methods.

Figure 2

Calcium ion coordination in the X-ray structures of S100B-Ca²⁺ in the presence and absence of TRTK-12. (a) X-ray crystal structure of S100B-Ca²⁺ (3IQO) shown in a ribbon diagram with two Ca²⁺ ions per subunit (cyan spheres) labeled I (EF1, Pseudo EF-hand) and II (EF2, Typical EF-hand) with the subunits of the symmetric S100B homodimer colored red and blue with alpha helices labeled (I–IV). (b) The positions for the sidechains of the Ca²⁺ coordinating residues are compared for the pseudo-EF-hand (Ser18, Glu21, Asp23, Lys26 and Glu31) and the canonical EF-hand (Asp61, Asp63, Asp65, Glu67, and Glu72) calcium-binding sites of S100B-Ca²⁺ (green) and S100B-Ca²⁺-TRTK12 (Magenta) (RMSD = 0.18 Å).

Figure 3

The X-ray and NMR structures of the S100B-Ca²⁺-TRTK12 complex. (a) Stereo view (walleye mode) of the TRTK-12 peptide when bound to S100B with the electron density maps calculated with the 2mF_o-DF_c coefficients (1.0σ) for TRTK-12. (b) Ribbon and surface diagram of the X-ray structure of S100B-Ca²⁺-TRTK12 (3IQQ) illustrating the location of TRTK-12 (green) together with residues of S100B that interact with the peptide (in yellow). (c) Ribbon and surface diagram of the NMR structure of S100B-Ca²⁺-TRTK12 (1MWN) illustrating the location of TRTK-12 (green) bound to rat S100B with residues colored (in yellow) that have intermolecular NOE correlations to TRTK-12.

Figure 4

Side-chains of S100B and TRTK-12 that are involved in the peptide-protein interface. (a) TRTK-12 peptide residues that are observed in the X-ray crystal structure are illustrated (T3-L11) highlighting residues on S100B that are within 4.0 Å of TRTK peptide (in boxes) and residues that are involved in hydrogen bonding in red. (b) View of TRTK-12 (green) and S100B (blue) illustrating side-chains involved in hydrophobic interactions (yellow) between the TRTK-12 and S100B. (c) View of TRTK-12 (green) and S100B (blue) illustrating side-chains involved in hydrogen bonds (dashed lines) between TRTK-12 and S100B. (d) The X-ray structure of the bovine S100B-Ca²⁺-TRTK12 complex (shown in blue and green) superposed on the NMR solution structure of the same complex with rat S100B (red PDB: 1MWN).

Figure 5

Changes in main and side-chain positioning for the two asymmetric units of S100B-Ca²⁺ (model A and B) upon binding TRTK-12. (a) The average RMSDs for the position of main-chain atoms for the two asymmetric units, Model A and Model B, were compared for the X-ray structure of S100B-Ca²⁺. (b) The average RMSDs for the position of side-chain atoms for the two asymmetric units, Model A and Model B, were compared next for the X-ray structure of S100B-Ca²⁺. (c) The average RMSDs for the position of main-chain atoms were compared for the X-ray structures of S100B-Ca²⁺ (model A) and S100B-Ca²⁺-TRTK12. (d) The average RMSDs for the position of side-chain atoms were compared for the X-ray structures S100B-Ca²⁺ (model A) and S100B-Ca²⁺-TRTK. (e) The average RMSDs for the position of main-chain atoms were compared for the X-ray structures of S100B-Ca²⁺ (model B) and S100B-Ca²⁺-TRTK12. (f) The average RMSDs for the position of side-chain atoms were compared for the X-ray structures of S100B-Ca²⁺ (model B) and S100B-Ca²⁺-TRTK. Those residues that have an average RMSD for main-chain and side-chain value greater than 0.4 and 1.0 Å, respectively, are labeled. Residues of S100B-Ca²⁺ that are affected by TRTK-12 binding are labeled in red.

Figure 6

Graph showing B-factor values for each residue in S100B-Ca²⁺ in the absence and presence of bound TRTK-12. (a) Average of main-chain atoms B-factor values per residue of S100B for S100B-Ca²⁺; model A (blue diamonds), S100B-Ca²⁺; model B (green squares), S100B-Ca²⁺-TRTK (red triangle). (inset) Average of main-chain atoms B-factor values per residues of S100B for S100B-Ca²⁺-pentamidine (orange pluses) and S100B-Ca²⁺-TRTK (red triangle). (b) Average of all atoms B-factors per residue of S100B for S100B-Ca²⁺; model A (blue diamonds), Ca²⁺-S100B; model B (green squares), S100B-Ca²⁺-TRTK (red triangle). (inset) Average of all atoms B-factors per residue of S100B for S100B-Ca²⁺-pentamidine (orange pluses) and S100B-Ca²⁺-TRTK (red triangle). (c) Ribbon diagram of the X-ray structure of S100B-Ca²⁺-TRTK (3IQQ) illustrating the residues of S100B (in red) that showed significant changes in B-factor values when the X-ray structural models for S100B-Ca²⁺-TRTK and S100B-Ca²⁺ (i.e. for both models A and B) were compared.