Full-contact domain labeling: identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein.
Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)P(2) suggested that, in addition to the inositol headgroup, a diacylglyceryl moiety was important for binding; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)P2 binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca(2+) decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding of PtdIns(4,5)P2 within two folded repeats of gelsolin has been calculated using these data.
Feng
L
L
Department of Medicinal Chemistry, The University of Utah, Salt Lake City, Utah 84112, USA.
Mejillano
M
M
Yin
H L
HL
Chen
J
J
Prestwich
G D
GD
D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D019281:Dimerization; D018260:Gelsolin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D019920:Photoaffinity Labels / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D000199:Actins; D018260:Gelsolin; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D019920:Photoaffinity Labels; D011108:Polymers; D002118:Calcium