The effects of CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography.
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.
Charpentier
Thomas H
TH
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.
Thompson
Laura E
LE
Liriano
Melissa A
MA
Varney
Kristen M
KM
Wilder
Paul T
PT
Pozharski
Edwin
E
Toth
Eric A
EA
Weber
David J
DJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D051347:CapZ Actin Capping Protein; D002417:Cattle; D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D051347:CapZ Actin Capping Protein; D046912:Multiprotein Complexes; D009414:Nerve Growth Factors; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; C475482:TRTK-12 peptide; D002118:Calcium