Capping protein binding to S100B: implications for the tentacle model for capping the actin filament barbed end.
S100B binds tightly to a 12-amino acid peptide derived from heterodimeric capping protein. In native intact capping protein, this sequence is in the C terminus of the alpha-subunit, which is important for capping the actin filament. This C-terminal region is proposed to act as a flexible "tentacle," extending away from the body of capping protein in order to bind actin. To this hypothesis, we analyzed the interaction between S100B and capping protein in solution. The C-terminal 28 amino acids of the alpha-subunit, the proposed tentacle, bound to S100B as a free synthetic peptide or a glutathione S-transferase fusion (K(d) approximately 0.4-1 microm). In contrast, S100B did not bind to whole native capping protein or functionally affect its capping activity. S100B does not bind, with any significant affinity, to the proposed alpha-tentacle sequence of whole native capping protein in solution. In the NMR structure of S100B complexed with the alpha-subunit-derived 12-amino acid peptide, the hydrophobic side of a short alpha-helix in the peptide, containing an important tryptophan residue, contacts S100B. In the x-ray structure of native capping protein, the corresponding sequence of the alpha-subunit C terminus, including Trp(271), interacts closely with the body of the protein. Therefore, our results suggest the alpha-subunit C terminus is not mobile as predicted by the tentacle model. Addition of non-ionic detergent allowed whole capping protein to bind weakly to S100B, indicating that the alpha-subunit C terminus can be mobilized from the surface of the capping protein molecule, presumably by weakening the hydrophobic binding at the contact site.
Wear
Martin A
MA
Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Cooper
John A
JA
D051339:Actin Depolymerizing Factors; D000818:Animals; D002645:Chickens; D018360:Crystallography, X-Ray; D051345:Destrin; D003902:Detergents; D057927:Hydrophobic and Hydrophilic Interactions; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry* / Q000378:metabolism*
D051339:Actin Depolymerizing Factors; D051345:Destrin; D003902:Detergents; D008840:Microfilament Proteins; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579158:S100b protein, rat