Dimerization of the Highly Conserved Light Chain Shared by Dynein and Myosin V*
- From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032-3305
Abstract
The M r 8,000 light chain originally identified in Chlamydomonas flagellar dynein is also a component of both cytoplasmic dynein and myosin V. Furthermore, this small protein has been implicated as an inhibitor of neuronal nitric oxide synthase, suggesting that it may play multiple regulatory roles within the cell. Covalent cross-linking of both dynein and myosin V using 1,5-difluoro-2,4-dinitrobenzene revealed that this light chain exists as a dimer in situ. This observation was confirmed using two additional amine-selective cross-linking reagents (dimethyl pimelimidate and disuccinimidyl suberate). When expressed as a C-terminal fusion with maltose-binding protein, the presence of the light chain caused the recombinant molecule to dimerize. Analysis of fusions containing truncated light chains identified the predicted amphiphilic helix (residues 14–32) as sufficient to cause dimerization; cross-linking required a second helical segment (residues 33–46). Together the data presented suggest that two light chains interact to form a parallel dimeric structure. This arrangement has significant implications for the potential functions of this highly conserved molecule and suggests a mechanism by which it might dissociate nitric oxide synthase.
Footnotes
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↵* This work was supported by a New Investigator Award from the Patrick and Catherine Weldon Donaghue Medical Research Foundation and by National Institutes of Health Grant GM 51293.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Biochemistry, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06032-3305. Tel.: 860-679-3347; Fax: 860-679-3408; E-mail: king{at}panda.uchc.edu.
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↵1 The abbreviations used are: LC, light chain; HC, heavy chain; IC, intermediate chain; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; DMP, dimethyl pimelimidate; DSS, disuccinimidyl suberate; MBP, maltose-binding protein; nNOS, neuronal nitric oxide synthase.
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↵2 S. E. Benashski, A. Harrison, and S. M. King, unpublished observations.
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↵3 The smallest LC within outer arm dynein from sperm of the sea urchin Strongylocentrotus purpuratus is specifically recognized by an antibody (R4058) made against theChlamydomonas M r 8,000 LC (S. M. King and A. G. Moss, unpublished observations).
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- Received February 7, 1997.
- Revision received June 17, 1997.
