Myosin V: regulation by calcium, calmodulin, and the tail domain

Calcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due to a compact conformation that is possible only with the whole molecule. The paradoxical finding that dFull moved actin in EGTA suggests that binding of the molecule to the substratum turns it on, perhaps mimicking cargo activation. Calcium slows, but does not stop the rate of actin movement if excess calmodulin (CaM) is present. Without excess CaM, calcium binding to the high affinity sites dissociates CaM and stops motility. We propose that a folded-to-extended conformational change that is controlled by calcium and CaM, and probably by cargo binding itself, regulates myosin V's ability to transport cargo in the cell

Myosin V exhibits a high duty cycle and large unitary displacement

Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin–like fragments of myosin V (M5HMM) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5HMM molecules indicates a duty cycle of ≥50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5HMM molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5HMM attachment to the motility surface or light chain content. The large M5HMM working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion

The R403Q Myosin Mutation Implicated in Familial Hypertrophic Cardiomyopathy Causes Disorder at the Actomyosin Interface

Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the beta-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype.Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow beta-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface.These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state

Two single-headed myosin V motors bound to a tetrameric adapter protein form a processive complex

The yeast class V myosin Myo4p moves processively in vivo in a cargo-dependent manner following formation of a double-headed complex with the adapter protein She3p and the mRNA-binding protein She2p

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ?-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state