The three-dimensional structure and subunit association of S100B in the apo and Ca(2+)-bound states, and its Ca(2+)-dependent interaction with target proteins

Abstract

S100B(beta beta), a member of the S100 protein family, is a Ca2+-binding protein with noncovalent interactions at its dimer interface. The solution structure of S100B(beta beta) has been determined, in the apo- and Ca2+-loaded state, using 2D, 3D, and 4D NMR spectroscopy. In both states, the S100beta subunits (91 residue) contain four alpha-helices and a small antiparallel beta-sheet which brings together the two helix-loop-helix Ca2+-binding domains (EF-hands). Both structures are found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with the high solubility of S100B(beta beta). The antiparallel alignment of helix 1 with 1 prime (other subunit) and of helix 4 with 4 prime, and the perpendicular association of these pairs of antiparallel helices forms an X-type four helical bundle at the dimer interface in both structures. However, the orientation of helix 3 relative to helices 1, 2, and 4 in each subunit of apo-S100B(beta beta) differs significantly from that of Ca2+-loaded S100B(beta beta). This conformational change is well illustrated by the difference in interhelical angle (delta omega=112 degrees) of the C-terminal EF-hands (apo-S100beta, omega=140 degrees; Ca2+-loaded S100beta, omega=106 degrees). For comparison, omega ranges from 118 degrees to 145 degrees in the apo-state and from 84 degrees to 128 degrees in the Ca2+-bound states of the EF-hands in calbindin D subscript 9k, calcyclin, and calmodulin. The significant conformational change required of C-terminal EF-hand for it to adopt the Ca2+-bound structure readily explains the dramatic spectral effects previously observed for S100B({beta beta) upon the addition of Ca2+. The conformational change exposes a cleft defined by residues in the loop linking the EF-hands, helix 3, and the C-terminal loop of Ca2+-loaded S100B({beta beta). This cleft is absent in the apo-structure and is therefore likely to play a role in target protein binding. S100B(beta beta) has been shown to interact with the tumor suppressor protein, p53, and this interaction was examined with a peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). Fluorescence and NMR spectroscopy experiments show that the p53 peptide binds to a location of S100B(beta beta) that probably involves residues in the hinge region (S41, L44, E45, E46) and the C-terminal loop (A83, C84, H85, E86, F87, F88) as previously predicted. However, residues in helix 3 (V52, V53, V56, T59) are also effected by p53 peptide binding, which supports the proposal that the conformational change observed in helix 3 plays a role in target protein binding. Finally, S100B(beta beta) was shown to exist (>99%) as a non-covalently associated dimer at concentrations as low as 1 nM (subunit concentration, 500 pM dimer) in apo and Ca2+-loaded state. Therefore, in reducing environments and at physiological concentrations, the noncovalent dimer is most likely the form of S100B presented to target proteins.