Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site.
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 Trp(501) in the relay loop.
Kovacs
Mihaly
M
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
Malnasi-Csizmadia
Andras
A
Woolley
Robert J
RJ
Bagshaw
Clive R
CR
D000225:Adenine / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016880:Anisotropy; D001665:Binding Sites; D004023:Dictyostelium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D024462:Myosin Type II / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D000251:Adenosine Triphosphatases; D024462:Myosin Type II; D000225:Adenine