title: Location of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis.
abstract: In addition to binding Ca(2+), the S100 protein S100B binds Zn(2+) with relatively high affinity as confirmed using isothermal titration calorimetry (ITC; K(d) = 94 +/- 17 nM). The Zn(2+)-binding site on Ca(2+)-bound S100B was examined further using NMR spectroscopy and site-directed mutagenesis. Specifically, ITC measurements of S100B mutants (helix 1, H15A and H25A; helix 4, C84A, H85A, and H90A) were found to bind Zn(2+) with lower affinity than wild-type S100B (from 2- to >25-fold). Thus, His-15, His-25, Cys-84, His-85, and perhaps His-90 of S100B are involved in coordinating Zn(2+), which was confirmed by NMR spectroscopy. Previous studies indicate that the binding of Zn(2+) enhances calcium and target protein-binding affinities, which may contribute to its biological function. Thus, chemical shift perturbations observed here for residues in both EF-hand domains of S100B during Zn(2+) titrations could be detecting structural changes in the Ca(2+)-binding domains of S100B that are pertinent to its increase in Ca(2+)-binding affinity in the presence of Zn(2+). Furthermore, Zn(2+) binding causes helix 4 to extend by one full turn when compared to Ca(2+)-bound S100B. This change in secondary structure likely contributes to the increased binding affinity that S100B has for target peptides (i.e., TRTK peptide) in the presence of Zn(2+).
authors: Molecular and Cell Biology Program, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201, USA.
authors: Paul T
authors: PT
authors: Wilder
authors: Donna M
authors: DM
authors: Baldisseri
authors: Ryan
authors: R
authors: Udan
authors: Kristen M
authors: KM
authors: Vallely
authors: David J
authors: DJ
authors: Weber
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D051381:Rats; 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*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579158:S100b protein, rat; D016159:Tumor Suppressor Protein p53; D015032:Zinc; D002118:Calcium