Solution Structure of Rat Apo-S100B(ββ) As Determined by NMR Spectroscopy,

Abstract

S100B(ββ), a member of the S100 protein family, is a Ca²⁺-binding protein with noncovalent interactions at its dimer interface. Each apo-S100β subunit (91 residues) has four α-helices and a small antiparallel β-sheet, consistent with two predicted helix−loop−helix Ca²⁺-binding domains known as EF-hands [Amburgey et al. (1995) J. Biomol. NMR 6, 171−179]. The three-dimensional solution structure of apo-S100B(ββ) from rat has been determined using 2672 distance (14.7 per residue) and 88 dihedral angle restraints derived from multidimensional nuclear magnetic resonance spectroscopy. Apo-S100B(ββ) is found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with its high solubility. At the symmetric dimer interface, 172 intermolecular nuclear Overhauser effect correlations (NOEs) define the antiparallel alignment of helix I with I‘ and of helix IV with IV‘. The perpendicular association of these pairs of antiparallel helices forms an X-type four-helical bundle at the dimer interface. Whereas, the four helices within each apo-S100β subunit adopt a unicornate-type four-helix bundle, with helix I protruding from the parallel bundle of helices II, III, and IV. Accordingly, the orientation of helix III relative to helices I, II, and IV in each subunit differs significantly from that known for other Ca²⁺-binding proteins. Indeed, the interhelical angle (Ω) observed in the C-terminal EF-hand of apo-S100β is −142°, whereas Ω ranges from 118° to 145° in the apo state and from 84° to 128° in the Ca²⁺-bound state for the EF-hands of calbindin D_9k, calcyclin, and calmodulin. Thus, a significant conformational change in the C-terminal EF-hand would be required for it to adopt a structure typical of the Ca²⁺-bound state, which could readily explain the dramatic spectral effects observed upon the addition of Ca²⁺ to apo-S100B(ββ).