Solution Structure of Zinc- and Calcium-Bound Rat S100B as Determined by Nuclear Magnetic Resonance Spectroscopy †,‡ Paul T. Wilder, § Kristen M. Varney, | Michele B. Weiss, § Rossitza K. Gitti, | and David J. Weber* ,§,| Molecular and Cell Biology Program, Department of Biochemistry and Molecular Biology, UniVersity of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201 ReceiVed NoVember 16, 2004; ReVised Manuscript ReceiVed February 21, 2005 ABSTRACT: The EF-hand calcium-binding protein S100B also binds one zinc ion per subunit with a relatively high affinity (K d ∼ 90 nM) [Wilder et al., (2003) Biochemistry 42, 13410-13421]. In this study, the structural characterization of zinc binding to calcium-loaded S100B was examined using high-resolution NMR techniques, including structural characterization of this complex in solution at atomic resolution. As with other S100 protein structures, the quaternary structure of Zn 2+ -Ca 2+ -bound S100B was found to be dimeric with helices H1, H1′, H4, and H4′ forming an X-type four-helix bundle at the dimer interface. NMR data together with mutational analyses are consistent with Zn 2+ coordination arising from His-15 and His-25 of one S100B subunit and from His-85 and Glu-89 of the other subunit. The addition of Zn 2+ was also found to extend helices H4 and H4′ three to four residues similar to what was previously observed with the binding of target proteins to S100B. Furthermore, a kink in helix 4 was observed in Zn 2+ -Ca 2+ - bound S100B that is not in Ca 2+ -bound S100B. These structural changes upon Zn 2+ -binding could explain the 5-fold increase in affinity that Zn 2+ -Ca 2+ -bound S100B has for peptide targets such as the TRTK peptide versus Ca 2+ -bound S100B. There are also changes in the relative positioning of the two EF-hand calcium-binding domains and the respective helices comprising these EF-hands. Changes in conformation such as these could contribute to the order of magnitude higher affinity that S100B has for calcium in the presence of Zn 2+ . S100B and other members of the S100 protein family have a conserved N-terminal S100 “pseudo” EF-hand and a C-terminal “typical” EF-hand Ca 2+ -binding motif (1, 2). Upon binding Ca 2+ , most dimeric S100 proteins undergo a conformational change that regulates their binding to target proteins as necessary for modulating numerous biological functions (3, 4). In addition, S100B and several other S100 proteins bind Zn 2+ in a site unique from the Ca 2+ -binding site (5). For example, Ca 2+ -bound S100B symmetrically binds two Zn 2+ per dimer (K d ) 94.2 ( 16.7 nM), with both zinc-binding sites having liganding residues contributed from each subunit at the dimer interface (6). While the biological function of Zn 2+ in S100 proteins is not completely understood, these divalent ions are not structural elements because the proteins are stable, folded, and active in Vitro in their absence. Instead, it is more likely that Zn 2+ plays a regulatory role by modulating the affinity of various S100 proteins for Ca 2+ and/or protein targets. In the case of S100B, the binding affinity of Ca 2+ increases by as much as 10-fold (7) and its affinity for a peptide derived from the protein CapZ (TRTK) 1 increases 5-fold (8) when Zn 2+ is present. Likewise, the Ca 2+ -dependent binding of peptides derived from giant phosphoprotein AHNAK to S100B is enhanced by the presence of Zn 2+ (9). Other S100 family members also bind Zn 2+ including S100A2 (K d ) 4.6 µM), S100A3 (K d ) 1.5 µM), S100A4 (affinity not reported), S100A5 ([Zn 2+ ] 0.5 ) 2 µM), S100A6 (K d ) 0.1 µM), and S100A7 (K d ) 100 µM) (10), and in each case, a conformational change is observed upon binding Zn 2+ ions. S100A12 binds Zn 2+ with relatively high affinity (K d < 10 nM) compared to other S100 proteins causing a large increase (∼1500-fold) in its affinity for Ca 2+ (11). The S100B/S100A1 heterodimer and S100A1 are also capable of binding Zn 2+ but with a lower affinity than the S100B homodimer (S100B > S100B/A1 > S100A1) (12). Nonethe- † This work was supported by grants from the National Institutes of Health (GM58888, to D.J.W.). ‡ Coordinates for the 20 best structures of rat Zn 2+ -Ca 2+ -S100B and the associated restraint files have been deposited in the Brookhaven Protein Data Bank under the PDB code 1XYD. * To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, Maryland 21201. Tele- phone: (410) 706-4354. Fax: (410) 706-0458. E-mail: dweber@ umaryland.edu. § Molecular and Cell Biology Program. | Department of Biochemistry and Molecular Biology. 1 Abbreviations: ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; TRTK, CapZR peptide ( 265 TRTKIDWNKILS 276 ); NDR, nuclear serine/threonine kinase peptide ( 62 KRLRRSAHARKET- EFLRLKRTRLGLE 87 ); ACS, American Chemical Society; p53 367-388 , human p53 peptide ( 367 SHLKSKKGQSTSRHKKLMFKTE 388 ); p53 F385W , fluorescent p53 peptide ( 367 SHLKSKKGQSTSRHKKLMWKTE 388 ); DTT, dithiothreitol; TES, (2-[(2-hydroxy-1,1-bis[hydroxymethyl]ethyl)- amino]ethanesulfonic acid); TPPI, time-proportional phase incremen- tation; TSP, 3-(trimethylsilyl)-propionic acid-D4, sodium salt; HSQC, heteronuclear single-quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; HOHAHA, homonuclear Hartman-Hahn spec- troscopy; HMQC, heteronuclear multiple-quantum coherence; DIPSI- 2, decoupling in the presence of scalar interaction version 2; EDTA, ethylenediaminetetraacetic acid; BMRB, BioMagResBank; rmsd, root- mean-square difference; PDB, Protein Data Bank; WT, wild type; 3D, three-dimensional; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylene- diamine. 5690 Biochemistry 2005, 44, 5690-5702 10.1021/bi0475830 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/22/2005