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Originally published In Press as doi:10.1074/jbc.M202180200 on April 23, 2002

J. Biol. Chem., Vol. 277, Issue 32, 28459-28467, August 9, 2002
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Analysis of Nucleotide Binding to Dictyostelium Myosin II Motor Domains Containing a Single Tryptophan Near the Active Site*

Mihály KovácsDagger §, András Málnási-CsizmadiaDagger §, Robert J. WoolleyDagger , and Clive R. BagshawDagger ||

From the Dagger  Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom and the § Department of Biochemistry, Eötvös Loránd University, Pázmány Péter Sétány 1/C, H-1117 Budapest, Hungary

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp501 in the relay loop.

* This work was supported by the BBSRC, Wellcome Trust, and the Magyary Zoltán Fellowship.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.

Supported by EMBO and the Hungarian Eötvös State Fellowship.

|| To whom correspondence should be addressed. Tel.: 44-116-252-3454; Fax: 44-116-252-3369; E-mail: crb5@le.ac.uk.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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