Susan Lowey

Evidence for cleft closure in actomyosin upon ADP release

Structural insights into the interaction of smooth muscle myosin with actin have been provided by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy, and by mapping of structural and dynamic changes in the actomyosin complex. The actomyosin structures determined in the presence and absence of MgADP differ significantly from each other, and from all crystallographic structures of unbound myosin. Coupled to a complex movement (∼34 Å) of the light chain binding domain upon MgADP release, we observed a ∼9° rotation of the myosin motor domain relative to the actin filament, and a closure of the cleft that divides the actin binding region of the myosin head. Cleft closure is achieved by a movement of the upper 50 kDa region, while parts of the lower 50 kDa region are stabilized through strong interactions with actin. This model supports a mechanism in which binding of MgATP at the active site opens the cleft and disrupts the interface, thereby releasing myosin from actin.

The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties.

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.

Functional Consequences of Mutations in the Myosin Heavy Chain at Sites Implicated in Familial Hypertrophic Cardiomyopathy

The primary cause of familial hypertrophic cardiomyopathy (FHC) has been attributed to mutations in the genes that encode the contractile proteins of the muscle cell. A majority of these mutations have been found in myosin, the principal component of the thick filament. Most in vitro studies have concluded that FHC mutations cause a loss of function in the biochemical and mechanical properties of myosin. Hypertrophy would then follow as a compensatory mechanism to raise the work and power output of the failing heart. Several recent studies, however, have thrown this mechanism into doubt by providing evidence that FHC mutations in the myosin heavy chain (MHC) can enhance the functional properties of myosin. This review discusses the problems encountered in reaching a definitive answer on the effect of MHC mutations.

Common structural motifs for the regulation of divergent class II myosins.

This minireview focuses on structural studies that have provided insights into our current understanding of thick filament regulation in muscle. We describe how different domains in the myosin molecule interact to produce an inactive “off” state; included are head-head and head-rod interactions, the role of the regulatory light chain, and the significance of the α-helical coiled-coil rod in regulation. Several of these interactions have now been visualized in a wide variety of native myosin filaments, testifying to the generality of these structural motifs across the phylogenetic tree.

Myosin isoforms show unique conformations in the actin-bound state

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, and more stable surface loops than those seen in the available crystal structures, indicating the existence of unique actin-bound conformations.

Crystal structures of monomeric actin bound to cytochalasin D.

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin have not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD: one obtained by soaking preformed actin crystals with CD, and the other obtained by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play equally important roles in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. An approximately 6 degrees rotation of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure instead of being disordered, as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.

The R403Q myosin mutation implicated in familial hypertrophic cardiomyopathy causes disorder at the actomyosin interface.

Background Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the β-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype. Principal Findings Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow β-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface. Significance These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state.

Transgenic mouse α- and β-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences.

Background: The mouse model for cardiac disease has been the focus for mutational studies on the α-cardiac myosin heavy chain (MHC). Results: Transient kinetics showed that R403Q enhanced ADP release in mouse α-MHC but not in mouse β-MHC. Conclusion: The functional impact of a mutation depends on the myosin isoform. Significance: The R403Q mutation in human β-cardiac myosin likely compromises its function leading to hypertrophic cardiomyopathy. Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μm) is ∼ 4-fold weaker than for β-S1 (25 μm). Correspondingly, the MgADP release rate for α-S1 (350 s−1) is ∼3-fold greater than for β-S1 (120 s−1). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s−1). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.

Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

Significance The separation of daughter cells during cell division, or cytokinesis, is a process that requires contractile rings which develop tension using actin and myosin. Current models of contractile ring dynamics are based on quantitative data from two decades of research using the tractable fission yeast system. However, it is unknown how fission yeast’s essential myosin, Myo2, is regulated in the contractile ring. Here, we find that Myo2 does not assemble into minifilaments, consistent with its role in ring precursor nodes. Unphosphorylated Myo2 exhibits robust enzymatic and motor activity whereas phosphorylation of Myo2’s regulatory light chain reduces its affinity for actin. This reduction likely weakens the tension in the contractile ring, potentially to delay cytokinesis until segregation of chromosomes is complete. Studies in fission yeast Schizosaccharomyces pombe have provided the basis for the most advanced models of the dynamics of the cytokinetic contractile ring. Myo2, a class-II myosin, is the major source of tension in the contractile ring, but how Myo2 is anchored and regulated to produce force is poorly understood. To enable more detailed biochemical/biophysical studies, Myo2 was expressed in the baculovirus/Sf9 insect cell system with its two native light chains, Rlc1 and Cdc4. Milligram yields of soluble, unphosphorylated Myo2 were obtained that exhibited high actin-activated ATPase activity and in vitro actin filament motility. The fission yeast specific chaperone Rng3 was thus not required for expression or activity. In contrast to nonmuscle myosins from animal cells that require phosphorylation of the regulatory light chain for activation, phosphorylation of Rlc1 markedly reduced the affinity of Myo2 for actin. Another unusual feature of Myo2 was that, unlike class-II myosins, which generally form bipolar filamentous structures, Myo2 showed no inclination to self-assemble at approximately physiological salt concentrations, as analyzed by sedimentation velocity ultracentrifugation. This lack of assembly supports the hypothesis that clusters of Myo2 depend on interactions at the cell cortex in structural units called nodes for force production during cytokinesis.