Modification of Cys-837 identifies an actin-binding site in the beta-propeller protein scruin.
In the acrosomal process of Limulus sperm, the beta-propeller protein scruin cross-links actin into a crystalline bundle. To confirm that scruin has the topology of a beta-propeller protein and to understand how scruin binds actin, we compared the solvent accessibility of cysteine residues in scruin and the acrosomal process by chemical modification with (1,5-IAEDANS). In soluble scruin, the two most reactive cysteines of soluble scruin are C837 and C900, whereas C146, C333, and C683 are moderately reactive. This pattern of reactivity is consistent with the topology of a typical beta-propeller protein; all of the reactive cysteines map to putative loops and turns whereas the unreactive cysteines lie within the predicted interior of the protein. The chemical reactivities of cysteine in the acrosomal process implicate C837 at an actin-binding site. In contrast to soluble scruin, in the acrosomal process, C837 is completely unreactive while the other cysteines become less reactive. Binding studies of chemically modified scruin correlate the extent of modification at C837 with the extent of inhibition of actin binding. Furthermore, peptides corresponding to residues flanking C837 bind actin and narrow a possible actin-binding region to a KQK sequence. On the basis of these studies, our results suggest that an actin-binding site lies in the C-terminal domain of scruin and involves a putative loop defined by C837.
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Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
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D000199:Actins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D006737:Horseshoe Crabs; D008297:Male; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013094:Spermatozoa / Q000737:chemistry; D013329:Structure-Activity Relationship; D017186:Titrimetry
D000199:Actins; D010455:Peptides; C083765:scruin protein, Horseshoe Crab; D004228:Dithionitrobenzoic Acid; D003545:Cysteine