Target binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand mutant proteins.

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TitleTarget binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand mutant proteins.
Publication TypeJournal Article
Year of Publication2012
AuthorsLiriano, MA, Varney, KM, Wright, NT, Hoffman, CL, Toth, EA, Ishima, R, Weber, DJ
JournalJ Mol Biol
Volume423
Issue3
Pagination365-85
Date Published2012 Oct 26
ISSN1089-8638
KeywordsBinding Sites, Calcium, Calcium-Binding Proteins, CapZ Actin Capping Protein, Crystallography, X-Ray, Mutant Proteins, Mutation, Nerve Growth Factors, Nuclear Magnetic Resonance, Biomolecular, Oligopeptides, Peptide Fragments, Protein Binding, Protein Conformation, S100 Calcium Binding Protein beta Subunit, S100 Proteins, Thermodynamics
Abstract

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca(2+) binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With (D63N)S100B as an exception ((D63N)K(D)=50±9 μM), Ca(2+) binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 ((D61N)K(D)=412±67 μM, (D65N)K(D)=968±171 μM, and (E72A)K(D)=471±133 μM), when compared to wild-type protein ((WT)K(D)=56±9 μM). For the TRTK-12 complexes, the Ca(2+)-binding affinity to wild type ((WT+TRTK)K(D)=12±10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target ((D61N+TRTK)K(D)=29±1.2 μM, (D63N+TRTK)K(D)=10±2.2 μM, (D65N+TRTK)K(D)=73±4.4 μM, and (E72A+TRTK)K(D)=18±3.7 μM). In addition, R(ex), as measured using relaxation dispersion for side-chain (15)N resonances of Asn63 ((D63N)S100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, (D61N)S100B, (D63N)S100B, and (D65N)S100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca(2+)-bound (2.0Å) and TRTK-bound (1.2Å) (D63N)S100B showed no change in Ca(2+) coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca(2+)-binding affinity in solution. Therefore, a model for how S100B-TRTK-12 complex formation increases Ca(2+) binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.

DOI10.1016/j.jmb.2012.07.011
Alternate JournalJ. Mol. Biol.
PubMed ID22824086
PubMed Central IDPMC3462298
Grant ListCA107331 / CA / NCI NIH HHS / United States
CA144560-02 / CA / NCI NIH HHS / United States
GM58888 / GM / NIGMS NIH HHS / United States
R01 CA107331 / CA / NCI NIH HHS / United States
R01 GM058888 / GM / NIGMS NIH HHS / United States
S10 RR015741 / RR / NCRR NIH HHS / United States
S10 RR016812 / RR / NCRR NIH HHS / United States
S10 RR023447 / RR / NCRR NIH HHS / United States
S10 RR029601 / RR / NCRR NIH HHS / United States
S10 RR031729 / RR / NCRR NIH HHS / United States
S10 RR10441 / RR / NCRR NIH HHS / United States
S10 RR15741 / RR / NCRR NIH HHS / United States
S10 RR16812 / RR / NCRR NIH HHS / United States
S10 RR23447 / RR / NCRR NIH HHS / United States