Structural rearrangements in the active site of smooth-muscle myosin.
Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 A and 24.9 A, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-A closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.
Robertson
C Ian
CI
Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, Vermont, USA.
Gaffney
Donald P
DP
Chrin
Lynn R
LR
Berger
Christopher L
CL
D020106:Acrylamide / Q000737:chemistry / Q000494:pharmacology; D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D024462:Myosin Type II / Q000737:chemistry; D009218:Myosins / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D018076:DNA, Complementary; D009711:Nucleotides; D020106:Acrylamide; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024462:Myosin Type II; D009218:Myosins