Regulation of Myosin V Processivity by Calcium at the Single Molecule Level

Abstract

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. Myosin V (myoV)2 is a processive motor capable of taking multiple steps on actin filaments before dissociation (1). A striking feature of the molecule is its extended neck region which binds 6 calmodulins (CaM) and acts as a lever arm. This long neck region enables myoV to take 36-nm steps along the actin filament through a hand-over-hand mechanism (2), mediated by strain between the two heads (reviewed in Ref. 3). In addition to the widely accepted mechanical role of the lever arm, it is likely that the neck may also be involved in the regulation of myoV activity, given that CaM is a universal calcium sensor. Each CaM binds to an "IQ motif," with a consensus sequence of IQXXXRGXXXR, where X denotes any amino acid.In the absence of calcium, the IQ motifs are fully occupied by CaM. In the presence of calcium, one or more CaM molecules dissociate from the neck (4 -6). Based on data obtained with truncated neck constructs, it is likely that calcium-CaM can dissociate from the second IQ motif relative to the motor domain (5, 7).The actin-activated ATPase activity of full-length myoV, but not of shorter dimeric constructs, is inhibited in the absence of calcium and activated in its presence. Accompanying the activity change is a conformational change from a folded (inhibited) to an extended (active) conformation, which is seen only with the full-length molecule (5,8,9). Recent three-dimensional reconstructions of crystalline arrays of the folded form of myoV revealed structural details of the inhibited state (10). In this model, the molecule bends at the junction of the sixth IQ motif and the ␣-helical coiled-coil, so that the cargo-binding globular tail docks onto the two motor domains. The site of interaction is near loop 1 at the entrance to the nucleotide binding pocket, thus providing a ...

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

“…Whereas low calcium concentrations activate the molecule (4-7), higher calcium levels inhibit motility (4,24) and terminate processive runs of single myosin V molecules (8,9). These results are consistent with the idea that calcium significantly weakens the affinity of CaM for one or more of the IQ motifs.…”

“…Under these conditions, myosin V adopts a folded, inactive conformation (4-7). Low calcium concentrations unfold and activate the molecule, whereas higher calcium concentrations inhibit motility and processive movement by dissociating CaM from one or more of the IQ motifs (4,8,9). Therefore, it is important to know the structure of CaM bound to the IQ motifs to understand more fully both the mechanical and regulatory properties of myosin V.…”

Effect of Calcium on Calmodulin Bound to the IQ Motifs of Myosin V

“…Once activated, higher calcium concentrations have an inhibitory effect on myosin V function; ensemble motility is inhibited, and processive runs are stopped (5,10,11). These effects are interpreted as due to dissociation of calcium-CaM from IQ motif 2.…”

“…Myosin V adopts a folded, inactive conformation under these conditions provided that cargo is not present (5)(6)(7)(8)(9). Low calcium concentrations or cargo binding in the absence of calcium unfold and activate the molecule, whereas higher calcium concentrations inhibit motility and processive movement by dissociating CaM from one or more of the IQ motifs (5,10,11).…”

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

“…S2 A-C). Such shifts in motor numbers may occur in vivo, for example, by local increases in calcium concentration in response to external stimuli, which would inhibit myoV processivity as observed in vitro (39,40). In addition, the actin tracks on which the motors travel, through actin-binding proteins, could differentially impact different classes of myosin motors.…”