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 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.

Complex Formation between S100B Protein and the p90 Ribosomal S6 Kinase (RSK) in Malignant Melanoma Is Calcium-dependent and Inhibits Extracellular Signal-regulated Kinase (ERK)-mediated Phosphorylation of RSK

“…However, another explanation is that the Ca 2ϩ binding affinity of S100 proteins can also be increased upon binding other metals and/or their physiologically relevant protein target(s). For example, it is well established in vitro that the affinity of S100B and other S100 proteins for Ca 2ϩ is increased after Zn 2ϩ binding (48,49), redox modification of critical cysteine residues (50), and/or by target binding, although the mechanism by which Ca 2ϩ binding is increased by as much as 200-fold upon target binding to S100B is still under investigation (18,(51)(52)(53).…”

Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2

“…Several structures of S100 proteins in complexes with their respective ligands have been published, including S100B in complex with a CapZ peptide (TRTK12; Charpentier et al, 2010), S100A11 in complex with the annexin A1 N-terminus (Ré ty et al, 2000) and S100A4 in complex with myosin IIA (Kiss et al, 2012). While these complexes generally have a quaternary structure similar to that of (p11) 2 (AnxA2) 2 , namely a peptide bound in each hydrophobic pocket of the activated S100 dimer (1:1 ratio of S100 protein:ligand), the structure of S100A4 in complex with a myosin IIA peptide was the first to reveal a asymmetric binding motif in which one peptide binds to the S100A4 dimer (1:2).…”

“…Therefore, the key hydrogen bonds adding to the stability of the complex made possible by the presence of AnxA2 should not exist. S100B does bind the CapZ peptide (TRTK12) and a crystal structure of the complex is available (PDB entry 3iqq; Charpentier et al, 2010). Superimposition of the S100B-CapZ structure onto AHNAK-(p11) 2 (AnxA2) 2 suggests that the hydrogen bond between the carbonyl of Gly1 (AHNAK) and the amide of Ser12 (AnxA2 chain D) could potentially occur with the amide of Asp6 (TRTK12) instead, as the backbone atoms of Ser12 (AnxA2) and Asp6 (TRTK12) are aligned.…”

The relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR data

“…Despite the issues discussed above, comparisons between B-factors and dynamic data obtained by NMR spectroscopy, are frequently discussed (Charpentier et al, 2010;Chevelkov et al, 2010;Clore & Schwieters, 2006;Liu et al, 2009;Yang et al, 2007;Yang, Tasayco et al, 2009), and in References 12-21, 28 from Clore and Schwieters (2006). In this contribution, we would like to discuss the question: How does true dynamic data describing molecular reorientations (as obtained by dynamic NMR) correlate with indirect dynamic data as displayed in X-ray B-factors?…”