Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport.

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (∼350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (∼200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface.

Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke.

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin's working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.

Calcium regulation of calmodulin binding to and dissociation from the myo1c regulatory domain.

Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calcium binding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but the kinetic details of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements, fluorescence spectroscopy, and stopped-flow kinetics to determine the biochemical parameters that define the calmodulin-regulatory-domain interaction. We found calcium moderately increases the actin-activated ATPase activity and completely inhibits actin gliding. Addition of exogenous calmodulin in the presence of calcium fully restores the actin gliding rate. A fluorescently labeled calmodulin mutant (N111C) binds to recombinant peptides containing the myo1c IQ motifs at a diffusion-limited rate in the presence and absence of calcium. Measurements of calmodulin dissociation from the IQ motifs in the absence of calcium show that the calmodulin bound to the IQ motif adjacent to the motor domain (IQ1) has the slowest dissociation rate (0.0007 s-1), and the IQ motif adjacent to the tail domain (IQ3) has the fastest dissociation rate (0.5 s-1). When the complex is equilibrated with calcium, calmodulin dissociates most rapidly from IQ1 (60 s-1). However, this increased rate of dissociation is limited by a slow calcium-induced conformational change (3 s-1). Fluorescence anisotropy decay of fluorescently labeled N111C bound to myo1c did not depend appreciably on Ca2+. Our data suggest that the calmodulin bound to the IQ motif adjacent to the motor domain is rapidly exchangeable in the presence of calcium and is responsible for regulation of myo1c ATPase and motile activity.

Kinetic characterization of the weak binding states of myosin V.

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.

The kinetic mechanism of Myo1e (human myosin-IC).

Myo1e is the widely expressed subclass-1 member of the myosin-I family. We performed a kinetic analysis of a truncated myo1e that consists of the motor and the single IQ motif with a bound calmodulin. We determined the rates and equilibrium constants for the key steps in the ATPase cycle. The maximum actin activated ATPase rate (V(max)) and the actin concentration at half-maximum of V(max) (K(ATPase)) of myo1e are similar to those of the native protein. The K(ATPase) is low (approximately 1 microm), however the affinity of myo1e for actin in the presence of ATP is very weak. A weak actin affinity and a rapid rate of phosphate release result in a pathway under in vitro assay conditions in which phosphate is released while myo1e is dissociated from actin. Actin activation of the ATPase activity and the low K(ATPase) are the result of actin activation of ADP release. We propose that myo1e is tuned to function in regions of high concentrations of cross-linked actin filaments. Additionally, we found that ADP release from actomyo1e is > 10-fold faster than other vertebrate myosin-I isoforms. We propose that subclass-1 myosin-Is are tuned for rapid sliding, whereas subclass-2 isoforms are tuned for tension maintenance or stress sensing.