Effect of Nucleotides and Actin on the Orientation of the Light Chain-Binding Domain in Myosin Subfragment 1

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Abstract
The X-ray structure of myosin head (S1) reveals the presence of a long α-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 α-helix (the “lever arm”) to produce ∼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 α-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 x and AlF4- but only 1.9 Å in the presence of vanadate; we do not interpret the 6 Å 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.