Structure of the shutdown state of myosin-2.

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Molecular mechanism regulating myosin and cardiac functions by ELC.

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67 pN/nm and 2.3 µm/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25 pN/nm and 1.7 µm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 µm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0 mmHg) were significantly higher than hearts from TgM(E56G) (66.2 mmHg) or C57/BL6 (59.3±3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1>hVLC-1(E56G)≈mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.

Shedding a little light on light chains.

Myosin II regulatory light chains have an important role in the organization and function of the contractile machinery at cytokinesis. Two recent reports provide new insights into these important proteins.

Characterization of myosin light chain in shrimp hemocytic phagocytosis.

Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp.

X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties.

Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using our system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, we prepared and crystallized stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain. We solved the x-ray crystal structure by molecular replacement and refined the resulting model against diffraction data to 2.2 Å resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.

Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle.

X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2(Delta2-46)) or disruption of the phosphorylation sites by substituting alanines (Dmlc2(S66A, S67A)) decreased the equatorial intensity ratio (I(20)/I(10)), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2(S66A, S67A)), but not N-terminal extension truncation (Dmlc2(Delta2-46)), decreased the 14.5nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2(S66A, S67A)) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2(Delta2-46; S66A, S67A)), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a "tether" to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain.

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings.

Recent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity.

A 29 residue region of the sarcomeric myosin rod is necessary for filament formation.

Myosin is a motor protein whose functional unit in the sarcomere is the thick filament. The myosin molecule is capable of self-assembly into thick filaments through its alpha-helical coiled-coil rod domain. To define more precisely the sequence requirements for this assembly, segments of the human fast IId skeletal myosin rod were expressed in Escherichia coli and examined differential solubility and the formation of ordered paracrystals. We show that both properties appear to require a 29 residue sequence (residues 1874 to 1902) near the C terminus of the rod region. To test further the role of this region in assembly, a protein was constructed which consisted of this assembly competence domain (ACD) fused to the carboxy terminus of an assembly-incompetent myosin rod fragment. This chimeric fragment exhibited myosin's characteristic solubility properties and formed ordered paracrystals. To complement these in vitro experiments, both a full-length myosin heavy chain (MYH) and one from which the 29 residues were deleted were transfected into cultured mammalian cells. While the full-length construct formed the spindle-shaped structures characteristic of arrays of thick filaments, the deleted MYH showed only diffuse staining throughout the cytoplasm by light microscopy. Thus, there appears to be a specific sequence in the C-terminal region of the myosin heavy chain rod which is necessary for ordered paracrystal formation and is sufficient to confer assembly properties to an assembly-incompetent rod fragment.

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain.

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.

Regulatory light chains of striated muscle myosin. Structure, function and malfunction.

Striated (skeletal and cardiac) muscle is activated by the binding of Ca(2+) to troponin C and is regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function is still not well understood. The N-terminal domain of RLC contains a 'Ca(2+)-Mg(2+)'-binding site and, analogous to that of smooth muscle myosin, also contains a phosphorylation site. During muscle contraction, the increase in Ca(2+) concentration activates the Ca(2+)/calmodulin-dependent myosin light chain kinase and leads to phosphorylation of the RLC. In agreement with other laboratories we have demonstrated that phosphorylation and Ca(2+) binding to the RLC play an important modulatory role in striated muscle contraction. Furthermore, the ventricular isoform of human cardiac RLC has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. Our recent studies have demonstrated that phosphorylation and Ca(2+) binding to human ventricular RLC are significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation, whether located in the proximity of the RLC 'Ca(2+)-Mg(2+)'-binding site or the phosphorylation site (Serine 15). We have also shown that there is a functional coupling between Ca(2+) and/or Mg(2+) binding to the RLC and phosphorylation and that the FHC mutations can affect this relationship. Further in vivo studies are necessary to investigate the mechanisms involved in the pathogenesis of RLC-linked FHC.

Multiple isoforms of myosin light chain 1 in pig diaphragm slow fibers: correlation with maximal shortening velocity and force generation.

Pig diaphragm slow fibers exhibit heterogeneity in myosin light chain 1 (MLC1) isoform expression, with many expressing fast-type MLC1 (MLC1F), as well as two isoforms of slow-type MLC1 (MLC1Sa and MLC1Sb). The goal of this study was to test if there is a relationship between MLC1 isoform expression and contractile properties among these fibers. Maximal shortening velocity (V(max)) and maximal isometric force generation, normalized with fiber cross-sectional area (P(o)/CSA), were measured in single fibers. V(max) was inversely related to the relative level of MLC1Sa. The level of MLC1Sa was reciprocally related to the levels of MLC1Sb and of MLC1F among individual fibers. Fibers expressing MLC1Sa and in which MLC1Sb was not detected generated greater P(o)/CSA, compared to fibers expressing MLC1Sb and not MLC1Sa. The results indicate a complex pattern of MLC1 isoform expression among pig diaphragm slow fibers and suggest that shortening velocity and force generation are modulated, in these fibers, by the MLC1 isoform composition.

Reconstitution of ventricular myosin with atrial light chains 1 improves its functional properties.

Atrial light chain 1 (ALC-1) is expressed in embryonic and hypertrophied human ventricles but not in normal adult human ventricles. We investigated the effects of recombinant human atrial light chains (hALC-1) on the structure and enzymatic activity of synthetic filaments of ventricular myosin. The endogenous ventricular myosin light chain 1 (VLC-1) was partially replaced by recombinant hALC-1 yielding hALC-1 levels of 12%, 24% and 42%. This reconstitution of ventricular myosin with hALC-1 did not change the length of synthetic myosin filaments but led to more rounded myosin heads in comparison with those of control filaments. Actin-activated ATPase activity of myosin, a parameter of functional activity of molecular motor, amounted to 79.5 nmol P(i)/mg per min in control myosin filaments. Reconstitution with hALC-1 caused a profound increase of the actin-activated myosin ATPase activity in a dose dependent manner, for example, synthetic myosin filaments formed with 12%, 24% and 42% hALC-1 reconstituted myosin revealed the actin-activated ATPase activity increased by 18%, 26% and 36%, respectively, as compared to control. These results strongly suggest that in vivo expression of ALC-1 enhances ventricular myosin function, thereby contributing to cardiac compensation.

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy.

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <<10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.

Fibre types in skeletal muscles of chronic obstructive pulmonary disease patients related to respiratory function and exercise tolerance.

This study aimed to investigate the relationship between skeletal muscle, fibre type composition, functional respiratory impairment and exercise tolerance in patients with moderate to severe chronic obstructive pulmonary disease (COPD). A group of 22 COPD patients and 10 healthy control subjects were studied. In COPD patients, vital capacity (VC) and forced expiratory volume in one second (FEV1) were reduced to 79% and 51%, respectively. Diffusion indices (transfer factor of the lung for carbon monoxide (TL,CO) and carbon monoxide transfer coefficient (KCO)) were also reduced. Arterial oxygen tension (Pa,O2) was normal or slightly altered. A maximal exercise test was performed and anaerobic threshold was calculated. Muscle samples from vastus lateralis were obtained by needle biopsy. Myosin heavy chain (MHC) and light chain (MLC) isoforms were separated by gel electrophoresis and quantified by densitometry. MHC isoforms were considered as molecular markers of fibre types. The proportion of the fast MHC-2B isoform was increased in COPD patients. TL,CO, KCO, VC and FEV1 were positively correlated with slow MHC isoform content. TL,CO and KCO were also negatively correlated with the content of the fast MHC-2B isoform. No correlation was found between exercise parameters and MHC isoform composition. The co-ordinated expression between MHC and MLC isoforms was altered in COPD patients. We conclude that reduced oxygen availability, probably in combination with muscle disuse, may determine muscle alterations in chronic obstructive pulmonary disease patients. The altered correlations between myosin heavy chain and light chain isoforms suggest that co-ordinated protein expression is lost in chronic obstructive pulmonary disease muscles.

Reorganization of myosin and focal adhesion proteins in Swiss 3T3 fibroblasts induced by transforming growth factor beta.

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF-beta). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell-adhesion molecules were redistributed in TGF-beta-treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western-blot analysis. Changes in cell morphology became apparent within 12 h of the addition of TGF-beta and new RNA and protein synthesis was necessitated by the changes. While TGF-beta induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF-beta treatment, with an increased amount. In addition, the cell-shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell-shape change, and acto-myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF-beta in Swiss 3T3 fibroblasts.

[The persistence of ontogenic characteristics in the adult masseter muscle]. / Persistance de caractères ontogéniques dans le muscle masséter adulte.

During embryonic and foetal development, the masseter is formed from two successive generations of muscle fibers in a manner which is very similar to that which has been previously described for other skeletal muscles. This phenotype is characterised by the persistence of ontogenic myosin isoforms (embryonic and foetal myosin heavy chains, embryonic light chain) and by the presence of two distinct populations of fibers: small diameter fibers which coexpress the embryonic, foetal and fast isoforms of the myosin heavy chains but never express the slow isoform; large diameter fibers which express the slow myosin heavy chain either exclusively or in variable associations with the other isoforms. These characteristics of the human masseter muscle probably correspond not only to its embryological origin and its special innervation, but also to the functional constraints to which it is submitted after birth.

Phosphorylation-dependent power output of transgenic flies: an integrated study.

We examine how the structure and function of indirect flight muscle (IFM) and the entire flight system of Drosophila melanogaster are affected by phosphorylation of the myosin regulatory light chain (MLC2). This integrated study uses site-directed mutagenesis to examine the relationship between removal of the myosin light chain kinase (MLCK) phosphorylation site, in vivo function of the flight system (flight tests, wing kinematics, metabolism, power output), isolated IFM fiber mechanics, MLC2 isoform pattern, and sarcomeric ultrastructure. The MLC2 mutants exhibit graded impairment of flight ability that correlates with a reduction in both IFM and flight system power output and a reduction in the constitutive level of MLC2 phosphorylation. The MLC2 mutants have wild-type IFM sarcomere and cross-bridge structures, ruling out obvious changes in the ultrastructure as the cause of the reduced performance. We describe a viscoelastic model of cross-bridge dynamics based on sinusoidal length perturbation analysis (Nyquist plots) of skinned IFM fibers. The sinusoidal analysis suggests the high power output of Drosophila IFM required for flight results from a phosphorylation-dependent recruitment of power-generating cross-bridges rather than a change in kinetics of the power generating step. The reduction in cross-bridge number appears to affect the way mutant flies generate flight forces of sufficient magnitude to keep them airborne. In two MLC2 mutant strains that exhibit a reduced IFM power output, flies appear to compensate by lowering wingbeat frequency and by elevating wingstroke amplitude (and presumably muscle strain). This behavioral alteration is not seen in another mutant strain in which the power output and estimated number of recruited cross-bridges is similar to that of wild type.

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.