Elena B. Krementsova

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 Vs ability to transport cargo in the cell.

Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can ‘walk’ hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5–7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head–S2 junction places the cargo-binding domain near the motor domains ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.

Myosin Va maneuvers through actin intersections and diffuses along microtubules

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.

Kinetic Characterization of a Monomeric Unconventional Myosin V Construct

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 ± 1.6 s−1 with aK app of ∼1 μm (37 °C), and the velocity of actin movement was ∼0.3 μm/s (30 °C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to ∼70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s−1), and ATP hydrolysis occurs at ∼200 s−1. The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.

Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features

A 2.5-Å resolution structure of calcium-free calmodulin (CaM) bound to the first two IQ motifs of the murine myosin V heavy chain reveals an unusual CaM conformation. The C-terminal lobe of each CaM adopts a semi-open conformation that grips the first part of the IQ motif (IQxxxR), whereas the N-terminal lobe adopts a closed conformation that interacts more weakly with the second part of the motif (GxxxR). Variable residues in the IQ motif play a critical role in determining the precise structure of the bound CaM, such that even the consensus residues of different motifs show unique interactions with CaM. This complex serves as a model for the lever arm region of many classes of unconventional myosins, as well as other IQ motif-containing proteins such as neuromodulin and IQGAPs.

The Light Chain Binding Domain of Expressed Smooth Muscle Heavy Meromyosin Acts as a Mechanical Lever

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or “neck.” To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.

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.

Tropomyosin Is Essential for Processive Movement of a Class V Myosin from Budding Yeast

Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class V myosins were characterized as nonprocessive in vitro, including Myo2p, the essential class V myosin from S. cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or behave processively in the cellular context. Here we show that Myo2p moves processively in vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.

A Nonprocessive Class V Myosin Drives Cargo Processively When a Kinesin- Related Protein Is a Passenger

During secretory events, kinesin transports cargo along microtubules and then shifts control to myosin V for delivery on actin filaments to the cell membrane [1]. When kinesin and myosin V are present on the same cargo, kinesin interacts electrostatically with actin to enhance myosin V-based transport in vitro [2]. The relevance of this observation within the cell was questioned. In budding yeast, overexpression of a kinesin-family protein (Smy1p) suppressed a transport defect in a strain with a mutant class V myosin (Myo2p) [3]. We postulate that this is a cellular manifestation of the in vitro observation. We demonstrate that Smy1p binds electrostatically to actin bundles. Although a single Myo2p cannot transport cargo along actin bundles, addition of Smy1p causes the complex to undergo long-range, continuous movement. We propose that the kinesin-family protein acts as a tether that prevents cargo dissociation from actin, allowing the myosin to take many steps before dissociating. We demonstrate that both the tether and the motor reside on moving secretory vesicles in yeast cells, a necessary feature for this mechanism to apply in vivo. The presence of both kinesin and myosin on the same cargo may be a general mechanism to enhance cellular transport in yeast and higher organisms.

Regulation of myosin V processivity by calcium at the single molecule level.

Calcium can affect myosin V (myoV) function in at least two ways. The full-length molecule, which adopts a folded inhibited conformation in EGTA, becomes extended and active in the presence of calcium. Calcium also dissociates one or more calmodulin molecules from the extended neck. Here we investigated at the single molecule level how calcium regulates the processive run length of full-length myosin V (dFull) and a truncated dimeric construct (dHMM), which cannot adopt the folded conformation. The processivity of dFull and dHMM is tightly controlled by the calcium and calmodulin concentration, with shorter runs occurring at higher calcium concentration. The data indicate that a calcium-dependent dissociation of calmodulin from the neck region of myoV terminates its processive run. dFull showed unexpected processive movement in EGTA, suggesting that a small population of extended, active molecules are in equilibrium with the inhibited, folded form. Single turnover assays showed that the ATPase activity of the folded full-length molecule is inhibited by more than 50-fold compared with the extended molecule. The results imply that activation and termination of the processive runs of myoV can be accomplished by multiple mechanisms.