Location of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis.

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TitleLocation of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis.
Publication TypeJournal Article
Year of Publication2003
AuthorsWilder, PT, Baldisseri, DM, Udan, R, Vallely, KM, Weber, DJ
Date Published2003 Nov 25
KeywordsAmino Acid Sequence, Amino Acid Substitution, Animals, Binding Sites, Calcium, Male, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Nerve Growth Factors, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Secondary, Rats, Recombinant Proteins, S100 Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Tumor Suppressor Protein p53, Zinc

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+).

Alternate JournalBiochemistry
PubMed ID14621986
Grant ListGM58888 / GM / NIGMS NIH HHS / United States
S10RR10441-01 / RR / NCRR NIH HHS / United States