A FRET-Based Sensor Reveals Large ATP Hydrolysis–Induced Conformational Changes and Three Distinct States of the Molecular Motor Myosin (original) (raw)
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A structural state of the myosin V motor without bound nucleotide
Nature, 2003
The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force 1 . Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core 1,2 , 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 Å 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.
Cell, 1998
The motor domain from the myosin II of the slime mold Dictyostelium discoideum crystallizes as a complex with a variety of bound nucleotides, many of which diffract to high resolution (reviewed in . The resulting structures fall into two classes that -9110 differ in the degree to which the cleft that splits the 50 kDa domain ("actin-binding cleft") is open and in the position of the so-called "converter" domain in the Summary C-terminal portion of the MD. Based on the geometry of the nucleotide in the binding pocket, the two con-The crystal structures of an expressed vertebrate formations have been identified as a prehydrolysis smooth muscle myosin motor domain (MD) and a mo-(MgADP·BeF x ) and a transition state (MgADP·AlF 4 Ϫ , tor domain-essential light chain (ELC) complex (MDE),
Myosin isoforms show unique conformations in the actin-bound state
Proceedings of the National Academy of Sciences, 2003
Crystallographic data for several myosin isoforms have provided evidence for at least two conformations in the absence of actin: a prehydrolysis state that is similar to the original nucleotide-free chicken skeletal subfragment-1 (S1) structure, and a transition-state structure that favors hydrolysis. These weak-binding states differ in the extent of closure of the cleft that divides the actin-binding region of the myosin and the position of the light chain binding domain or lever arm that is believed to be associated with force generation. Previously, we provided insights into the interaction of smooth-muscle S1 with actin by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy. Here, we analyze the conformations of actin-bound chicken skeletal muscle S1. We conclude that both myosin isoforms in the nucleotide-free, actin-bound state can achieve a more tightly closed cleft, a more downward position of the lever arm,...
Mechanics of actomyosin bonds in different nucleotide states are tuned to muscle contraction
2006
Muscle contraction and many other cell movements are driven by cyclic interactions between actin filaments and the motor enzyme myosin. Conformational changes in the actin-myosin binding interface occur in concert with the binding of ATP, binding to actin, and loss of hydrolytic by-products, but the effects of these conformational changes on the strength of the actomyosin bond are unknown. The force-dependent kinetics of the actomyosin bond may be particularly important at high loads, where myosin may detach from actin before achieving its full power stroke. Here we show that over a physiological range of rapidly applied loads, actomyosin behaves as a ''catch'' bond, characterized by increasing lifetimes with increasing loads up to a maximum at Ϸ6 pN. Surprisingly, we found that the myosin-ADP bond is possessed of longer lifetimes under load than rigor bonds, although the load at which bond lifetime is maximal remains unchanged. We also found that actomyosin bond lifetime is ultimately dependent not only on load, but loading history as well. These data suggest a complex relationship between the rate of actomyosin dissociation and muscle force and shortening velocity. The 6-pN load for maximum bond lifetime is near the force generated by a single myosin molecule during isometric contraction. This raises the possibility that all catch bonds between load-bearing molecules are ''mechanokinetically'' tuned to their physiological environment. catch bonds ͉ dynamic force spectroscopy ͉ laser trap ͉ myosin T he crystal structure of the myosin head (S1) (1) reveals how small-scale conformational changes within the hydrolytic site are converted into relatively large-scale changes producing movement. A globular ''motor domain'' occupies the bulk of the structure and contains both the nucleotide-and actin-binding sites (see Fig. 1). Of particular note is an ␣-helical extension of the heavy chain, or ''neck,'' protruding from the globular motor domain that acts as a rigid ''lever arm'' to amplify small movements arising in the nucleotide-binding pocket (2-5). Concomitant with phosphate release, rotation of the neck causes a step of an Ϸ5.5-nm ''working stroke'' and an isometric force of 0.7-9 pN (3, 6-10) that has been the subject of numerous single-molecule mechanics studies. Another feature of particular note within the motor domain is a cleft that divides the actin-binding site (2). This cleft is thought to close upon binding to actin (1, 2, 11-13), bringing into position residues on both sides of the cleft that are involved in strong binding to actin: the so-called R-site and A-site. Evidence suggests that there may be additional conformational changes in the actin-binding interface that accompany ADP release. The cleft at the actin-binding interface of smooth muscle myosin may close further upon ADP release (14, 15) and is accompanied by an increase in R-site flexibility (14, 16). Thus, although both the R-site and A-site are tightly bound to actin in the presence of ADP, upon ADP release from smooth muscle myosin the R-site adopts a more flexible conformation whereas the A-site remains tightly bound. EM reconstructions reveal no similar closure of the cleft in skeletal muscle myosin upon ADP release, although the resolution of the technique was limited. It does appear, however, that there are increases in R-site flexibility in skeletal muscle myosin upon ADP release (14).
Journal of Biological Chemistry, 2004
Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of ϳ30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was ϳ60 nm, strongly supporting a handover hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position,whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin.
Proceedings of the National Academy of Sciences, 2014
Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/ extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s −1 ) is similar to the maximum ATPase activity (∼12-14 s −1 ) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load , myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircaselike processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.