title: Identification of calcium-independent and calcium-enhanced binding between S100B and the dopamine D2 receptor.
abstract: S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100-300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. (1)H-(15)N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca(2+)-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca(2+)-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Dempsey
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism*; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation / Q000502:physiology
chemical_list: D001059:Apoproteins; D009414:Nerve Growth Factors; D017448:Receptors, Dopamine D2; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; D002118:Calcium