Characterization of the actin cross-linking properties of the scruin-calmodulin complex from the acrosomal process of Limulus sperm.
During activation of the Limulus sperm acrosomal process, actin filaments undergo a change in twist that is linked with the conversion from a coiled to a straight scruin-actin bundle. Since scruin had not been purified, its identity as an actin-binding protein has not been demonstrated. Using HECAMEG (methyl-6-O-(N-heptyl-carbamoyl)-alpha-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified it as an equimolar complex with calmodulin. 125I-Calmodulin overlays and calmodulin-Sepharose indicate that scruin binds calmodulin in calcium but not in EGTA. Overlay experiments also map the calmodulin binding site between the putative N- and C-terminal beta-propeller domains within residues 425-446. Immunofluorescence microscopy reveals that calmodulin colocalizes with scruin and actin in the coiled bundle. Although scruin binds calmodulin, pelleting assays and electron microscopy show that the scruin cross-links F-actin into bundles independently of calcium. Based on our biochemical and structural studies, we suggest a model to explain how scruin controls a change in twist of actin filaments during the acrosome reaction. We predict that calcium subtly alters scruin conformation through its calmodulin subunit and the conformation change in scruin causes a shift in the relative positions of the scruin-bound actin subunits.
Sanders
M C
MC
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02145, USA.
Way
M
M
Sakai
J
J
Matsudaira
P
P
D000177:Acrosome / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D006737:Horseshoe Crabs; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011817:Rabbits; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D002147:Calmodulin; C083765:scruin protein, Horseshoe Crab