Characterization of the Actin Cross-linking Properties of the Scruin-Calmodulin Complex from the Acrosomal Process of Limulus Sperm (*)
- From the (1)Whitehead Institute for Biomedical Research and the
- (2)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142
- §To whom all correspondence should be addressed: Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02145. Tel.: 617-258-5423; Fax: 617-258-7663.
Abstract
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-heptylcarbamoyl)-α-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified
it as an equimolar complex with calmodulin. 
I-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 β-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.
Footnotes
-
↵* This work was supported by National Institutes of Health Grants CA55621 and GM52703 (to P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- EM
-
electron microscopy
- HECAMEG
-
methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside
- HPLC
-
high performance liquid chromatography
- PAGE
-
polymacrylamide gel electrophoresis
- GST
-
glutathione S-transferase.
-
- Received July 11, 1995.
- Revision received October 5, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
