title: Target binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand mutant proteins.
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.
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors: Melissa A
authors: MA
authors: Liriano
authors: Kristen M
authors: KM
authors: Varney
authors: Nathan T
authors: NT
authors: Wright
authors: Cassandra L
authors: CL
authors: Hoffman
authors: Eric A
authors: EA
authors: Toth
authors: Rieko
authors: R
authors: Ishima
authors: David J
authors: DJ
authors: Weber
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051347:CapZ Actin Capping Protein; D018360:Crystallography, X-Ray; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D002135:Calcium-Binding Proteins; D051347:CapZ Actin Capping Protein; D050505:Mutant Proteins; D009414:Nerve Growth Factors; D009842:Oligopeptides; D010446:Peptide Fragments; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C475482:TRTK-12 peptide; D002118:Calcium