The crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE), both with a transition state analog (MgADP x AIF4-) in the active site, have been determined to 2.9 A and 3.5 A resolution, respectively. The MDE structure with an ATP analog (MgADP x BeFx) was also determined to 3.6 A resolution. In all three structures, a domain of the C-terminal region, the "converter," is rotated approximately 70 degrees from that in nucleotide-free skeletal subfragment 1 (S1). We have found that the MDE-BeFx and MDE-AIF4- structures are almost identical, consistent with the fact that they both bind weakly to actin. A comparison of the lever arm positions in MDE-AIF4- and in nucleotide-free skeletal S1 shows that a potential displacement of approximately 10 nm can be achieved during the power stroke.

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We have determined the structure of the intact scallop myosin head, containing both the motor domain and the lever arm, in the nucleotide-free state and in the presence of MgADP⅐VO4, corresponding to the transition state. These two new structures, together with the previously determined structure of scallop S1 complexed with MgADP (which we interpret as a detached ATP state), reveal three conformations of an intact S1 obtained from a single isoform. These studies, together with new crystallization results, show how the conformation of the motor depends on the nucleotide content of the active site. The resolution of the two new structures (Ϸ4 Å) is sufficient to establish the relative positions of the subdomains and the overall conformation of the joints within the motor domain as well as the position of the lever arm. Comparison of available crystal structures from different myosin isoforms and truncated constructs in either the nucleotide-free or transition states indicates that the major features within the motor domain are relatively invariant in both these states. In contrast, the position of the lever arm varies significantly between different isoforms. These results indicate that the heavy-chain helix is pliant at the junction between the converter and the lever arm and that factors other than the precise position of the converter can influence the position of the lever arm. It is possible that this pliant junction in the myosin head contributes to the compliance known to be present in the crossbridge.R ecent results on the myosin motor are beginning to reveal how this machine transforms chemical energy into movement. Crystallographic and biochemical studies on a number of isoforms (1-6) have identified at least three conformational states of the motor, which appear to correspond to different steps in the actomyosin cycle. The first crystal structure determined was that of methylated chicken skeletal muscle S1 without nucleotide (which contains a SO 4 2Ϫ ion in the active site; ref. 1). Although not bound to actin, this structure was considered to correspond closely to the postpower stroke (or rigor state), because the lever arm is in the ''down'' position (at an angle of Ϸ45°to the actin filament axis). This remarkable structure also led to the concept that the movement of the light-chain-bearing lever arm is responsible for force production and motility. Next, the structures of an expressed Dictyostelium motor domain (MD; lacking the lever arm) complexed with various nucleotide analogs were determined-often at high resolution (2-4). This work revealed detailed images of the nucleotide-binding site and led to the concept of myosin as a ''back door'' enzyme. The truncated structures fell into two classes, which were interpreted differently by Gulick and Rayment (7) and by Holmes (8). The latter author modeled one class of structures as corresponding to the ''primed'' or transition state, where the lever arm (if present) would be in an ''up'' position (at Ϸ90°to the actin filament axis), and the other class...

The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.

The crystal structure at 2.0-Å resolution of an 81-residue N-terminal fragment of muscle ␣-tropomyosin reveals a parallel two-stranded ␣-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (Ϸ1.2-Å) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded ␣-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.