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    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

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    Author
    Drohat, Alexander Clark
    Advisor
    Weber, David J., Ph.D.
    Date
    1997
    Type
    dissertation
    
    Metadata
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    Other Titles
    The three-dimensional structure and subunit association of S100B in the apo- and Ca2+-loaded states, and its Ca2+-dependent interaction with target proteins
    The three-dimensional structure and subunit association of S100B in the apo and Ca2+-bound states, and its Ca2+-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.
    Description
    University of Maryland, Baltimore. Biochemistry and Molecular Biology. Ph.D. 1997
    Keyword
    Biology, Molecular
    Chemistry, Biochemistry
    Biophysics, General
    S100B
    S100 Calcium Binding Protein beta Subunit
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1406
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