title: Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1.
abstract: The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to produce approximately 11 nm steps. To probe the spatial position of the putative lever in various S1 states, we have measured, by fluorescence resonance energy transfer (FRET), the effect of nucleotides and actin on the distances between Cys-177 of the essential light chain A1 (which is attached to the alpha-helix) and three loci in the catalytic domain. Cys-177 (donor) was labeled with 1,5-IAEDANS. The trinitrophenylated ADP analog (TNP-ADP, acceptor) was used to measure the distance to the active site. Lys-553 at the actin-binding site, labeled with a fluorescein derivative, and Lys-83 modified with trinitrobenzenesulfonic acid served as two other acceptors. FRET measurements were performed for S1 alone, for its complexes with MgADP and MgATP, for the analogs of the transition state of the ATPase reaction, S1.ADP.BeFx, S1.ADP.AlF4, and S1.ADP.VO4, and for acto-S1 in the absence and in the presence of ADP. When the transition state and acto-S1 complexes were formed, the change in the Cys-177 --> Lys-83 distance was <1.1 A, for the distance Cys-177 --> Lys-553, the change was +/-2.5 A. These distance changes correspond to rotations by <10 degrees and approximately 25 degrees, respectively. For the Cys-177 --> TNP-ADP the interprobe separation decreased by approximately 6 A in the presence of BeFx and AlF4- but only 1.9 A in the presence of vanadate; we do not interpret the 6 A change as resulting from the lever rotation. Using the coordinates of the acto-S1 complex, we have computed the expected changes in these distances resulting from rotation of the lever. These changes were much greater than the ones observed. The above results are inconsistent with models of force generation by S1 in which the head assumes two distinct conformations characterized by large differences in the angle between the motor and the light chain-binding domain. Several alternative mechanisms are proposed.
authors.affiliation: Centre de Recherches de Biochimie Macromoléculaire, CNRS, BP 5051, 34033 Montpellier Cedex, France.
authors.forename: C
authors.initials: C
authors.lastname: Smyczynski
authors.forename: A A
authors.initials: AA
authors.lastname: Kasprzak
mesh_terms: D000199:Actins / Q000494:pharmacology*; D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D018360:Crystallography, X-Ray; D004735:Energy Transfer; D018994:Myosin Light Chains / Q000737:chemistry* / Q000187:drug effects; D015879:Myosin Subfragments / Q000737:chemistry* / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D000199:Actins; D000227:Adenine Nucleotides; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate