The C-terminus and linker region of S100B exert dual control on protein-protein interactions with TRTK-12.
S100B, an EF-hand calcium-binding protein composed of two S100beta monomers, undergoes a calcium-dependent conformational change that provides a surface for target interactions. In this study, the calcium-sensitive S100B-binding epitope TRTK-12 has been used to probe the contributions of the linker and C-terminal regions of S100B to protein-protein interactions. These contributions were quantified using C-terminal mutant S100B proteins lacking the C-terminal seven (S100B85stop) or nine (S100B83stop) residues or containing alanine substitutions at Phe87 (F87A), Phe88 (F88A), or both (F8788A). Both F8788A and F88A bound TRTK-12 less tightly (K(d) = 1.85 +/- 0.02 and 0.97 +/- 0.08 microM, respectively) than the wild-type protein (K(d) = 0.27 +/- 0.03 microM, DeltaG = -37.2 kJ/mol), indicating these residues are important for TRTK-12 interaction. The truncated S100B proteins bound TRTK-12 much more weakly (K(d) = 659.7 +/- 119.3 microM, DeltaG = -17.9 kJ/mol), indicating the linker region contributed about 50% to the binding of TRTK-12, while the C-terminus contributed the remaining 50% of the binding energy. Based on mutagenesis and NMR chemical shift studies, a comparison with known S100-target protein complexes showed the S100B-TRTK-12 complex has the strongest resemblance to the S100A10-annexin II interaction.
McClintock
Kimberly A
KA
Department of Biochemistry and McLaughlin Macromolecular Structure Facility, The University of Western Ontario, London, Ontario, Canada.
Van Eldik
Linda J
LJ
Shaw
Gary S
GS
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011522:Protons; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002135:Calcium-Binding Proteins; D009414:Nerve Growth Factors; D009842:Oligopeptides; D010446:Peptide Fragments; D011522:Protons; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; D010649:Phenylalanine