The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force. Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core, the structural details of the rearrangement in myosin that drive the lever arm motion on actin attachment are unknown. Motivated by kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we obtained a 2.0 A structure of a myosin V fragment. Here we reveal a conformation of myosin without bound nucleotide. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule, and reveal elements of the structural communication between the actin-binding interface and nucleotide-binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

No abstract

We have solved a 2.4Å structure of a truncated version of the reverse direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulins. The structure reveals only minor differences in the motor domain as compared to plus-end directed myosins, with the exception of two unique inserts. The first insert is near the nucleotide-binding pocket, and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a previously unseen binding motif within the insert. This serves to redirect the effective "lever arm" of myosin VI, which includes a second calmodulin bound to an "IQ motif," towards the pointed (−) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to fully explain the movements of myosin VI.The myosin superfamily is composed of eighteen classes of molecular motor proteins, the vast majority of which traffic toward the barbed (+) end of actin filaments 1 . Class VI myosins were the first of the superfamily identified to traffic toward the pointed (−) end of the actin filament 2 . They function in a number of critical intracellular processes such as vesicular membrane traffic, cell migration, maintenance of stereocilia and mitosis [3][4][5][6] .The current view of how myosin motors couple ATP hydrolysis and actin binding to movement is known as the lever arm hypothesis 7 . In essence the proposed mechanism is that nucleotide binding, hydrolysis and product release are all coupled to small movements within the myosin motor core. These movements are amplified and transmitted via a region that has been termed the "converter" domain to a lever arm consisting of a target helix and associated light chains/ calmodulins. The lever arm further amplifies the motions of the converter domain into large directed movements. Consistent with the lever arm hypothesis, the stroke size has been shown to be proportional to the lever arm length 8,9,10 . In the absence of actin, ATP hydrolysis occurs, but product release is slow, thus trapping the lever arm in a primed or pre-powerstroke position.Correspondence should be addressed to either: Anne Houdusse, Structural Motility, UMR 144 Institut Curie-CNRS, 26 rue d'ULM, 75248 Paris cedex 05, France,, Anne.Houdusse@curie.fr or H. Lee Sweeney, Dept. of Physiology, University of Penn., A700 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6085, Tel. 215-898-8727, Fax. 215-573-2273, Lsweeney@mail.med.upenn.edu Binding to actin causes release of products, plus-end-directed movement of the lever arm, and force generation concomitant with formation of strong binding between myosin and actin.Perhaps because of their reverse directionality, myosin VI motors have a number of additional unusual features. First, the motor domain itself contains two inserts that are unique within...

The small molecule drug omecamtiv mecarbil (OM) specifically targets cardiac muscle myosin and is known to enhance cardiac muscle performance, yet its impact on human cardiac myosin motor function is unclear. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2β-S1 is slowed more than 4-fold in the presence of OM, whereas the actin concentration required for half-maximal ATPase was reduced dramatically (30-fold). We find OM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence of OM. The dominant pathway results in a slow transition between actomyosin·ADP states and increases the time myosin is strongly bound to actin. However, OM also traps a population of myosin heads in a weak actin affinity state with slow product release. We demonstrate that OM can reduce the actin sliding velocity more than 100-fold in the motility assay. The ionic strength dependence of motility suggests the inhibition may be at least partially due to drag forces from weakly attached myosin heads. OM causes an increase in duty ratio examined in the motility assay. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force development () in a concentration-dependent manner, whereas the maximally activated force is unchanged. We propose that OM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium.