A weak coupling mechanism for the early steps of the recovery stroke of myosin VI: A free energy simulation and string method analysis.

Myosin motors use the energy of ATP to produce force and directed movement on actin by a swing of the lever-arm. ATP is hydrolysed during the off-actin re-priming transition termed recovery stroke. To provide an understanding of chemo-mechanical transduction by myosin, it is critical to determine how the reverse swing of the lever-arm and ATP hydrolysis are coupled. Previous studies concluded that the recovery stroke of myosin II is initiated by closure of the Switch II loop in the nucleotide-binding site. Recently, we proposed that the recovery stroke of myosin VI starts with the spontaneous re-priming of the converter domain to a putative pre-transition state (PTS) intermediate that precedes Switch II closing and ATPase activation. Here, we investigate the transition from the pre-recovery, post-rigor (PR) state to PTS in myosin VI using geometric free energy simulations and the string method. First, our calculations rediscover the PTS state agnostically and show that it is accessible from PR via a low free energy transition path. Second, separate path calculations using the string method illuminate the mechanism of the PR to PTS transition with atomic resolution. In this mechanism, the initiating event is a large movement of the converter/lever-arm region that triggers rearrangements in the Relay-SH1 region and the formation of the kink in the Relay helix with no coupling to the active site. Analysis of the free-energy barriers along the path suggests that the converter-initiated mechanism is much faster than the one initiated by Switch II closure, which supports the biological relevance of PTS as a major on-pathway intermediate of the recovery stroke in myosin VI. Our analysis suggests that lever-arm re-priming and ATP hydrolysis are only weakly coupled, so that the myosin recovery stroke is initiated by thermal fluctuations and stabilised by nucleotide consumption via a ratchet-like mechanism.

Myosin VI rewrites the rules for myosin motors.

Myosin VI is the only type of myosin motor known to move toward the minus ends of actin filaments. This reversal in the direction of its movement is in part a consequence of the repositioning of its lever arm. In addition, myosin VI has a number of other specialized structural and functional adaptations that optimize performance of its unique cellular roles. Given that other classes of myosins may share some of these features, understanding the design principles of myosin VI will help guide the study of the functions of myosins that adopt similar strategies.

Switch between large hand-over-hand and small inchworm-like steps in myosin VI.

Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities.

Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.

Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

Myosin VI undergoes cargo-mediated dimerization.

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. We report the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and show that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X.

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart.

Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, ß-myosin heavy chain (ß-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike ß-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of ß-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate ß-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition.

Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization.

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 µM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis.

The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual approximately 180 degrees lever arm swing and achieves its large step size using special structural features in its tail domain.

Cellular defects resulting from disease-related myosin II mutations in Drosophila.

The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. Here we use the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, we show that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.

Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain.

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal ß-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Non-uniform distribution of myosin-mediated forces governs red blood cell membrane curvature through tension modulation.

The biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types.

An intermediate along the recovery stroke of myosin VI revealed by X-ray crystallography and molecular dynamics.

Myosins form a class of actin-based, ATPase motor proteins that mediate important cellular functions such as cargo transport and cell motility. Their functional cycle involves two large-scale swings of the lever arm: the force-generating powerstroke, which takes place on actin, and the recovery stroke during which the lever arm is reprimed into an armed configuration. Previous analyses of the prerecovery (postrigor) and postrecovery (prepowerstroke) states predicted that closure of switch II in the ATP binding site precedes the movement of the converter and the lever arm. Here, we report on a crystal structure of myosin VI, called pretransition state (PTS), which was solved at 2.2 Å resolution. Structural analysis and all-atom molecular dynamics simulations are consistent with PTS being an intermediate along the recovery stroke, where the Relay/SH1 elements adopt a postrecovery conformation, and switch II remains open. In this state, the converter appears to be largely uncoupled from the motor domain and explores an ensemble of partially reprimed configurations through extensive, reversible fluctuations. Moreover, we found that the free energy cost of hydrogen-bonding switch II to ATP is lowered by more than 10 kcal/mol compared with the prerecovery state. These results support the conclusion that closing of switch II does not initiate the recovery stroke transition in myosin VI. Rather, they suggest a mechanism in which lever arm repriming would be mostly driven by thermal fluctuations and eventually stabilized by the switch II interaction with the nucleotide in a ratchet-like fashion.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Structural mechanism for versatile cargo recognition by the yeast class V myosin Myo2.

Class V myosins are actin-dependent motors, which recognize numerous cellular cargos mainly via the C-terminal globular tail domain (GTD). Myo2, a yeast class V myosin, can transport a broad range of organelles. However, little is known about the capacity of Myo2-GTD to recognize such a diverse array of cargos specifically at the molecular level. Here, we solved crystal structures of Myo2-GTD (at 1.9-3.1 Å resolutions) in complex with three cargo adaptor proteins: Smy1 (for polarization of secretory vesicles), Inp2 (for peroxisome transport), and Mmr1 (for mitochondria transport). The structures of Smy1- and Inp2-bound Myo2-GTD, along with site-directed mutagenesis experiments, revealed a binding site in subdomain-I having a hydrophobic groove with high flexibility enabling Myo2-GTD to accommodate different protein sequences. The Myo2-GTD-Mmr1 complex structure confirmed and complemented a previously identified mitochondrion/vacuole-specific binding region. Moreover, differences between the conformations and locations of cargo-binding sites identified here for Myo2 and those reported for mammalian MyoVA (MyoVA) suggest that class V myosins potentially have co-evolved with their specific cargos. Our structural and biochemical analysis not only uncovers a molecular mechanism that explains the diverse cargo recognition by Myo2-GTD, but also provides structural information useful for future functional studies of class V myosins in cargo transport.

PTP1B up-regulates EGFR expression by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. Protein tyrosine phosphatase 1B (PTP1B) is a member of protein tyrosine phosphatases (PTPs) family. In our previous work, PTP1B was found to be overexpressed in ESCC tissues and made contributions to the the cell migration and invasion as well as lung metastasis of ESCC. In this study, we explored the underlying molecular mechanisms. PTP1B enhanced cell migration and invasion by promoting epidermal growth factor receptor (EGFR) expression in ESCC, which was relied on phosphatase activity of PTP1B. Using GST-pulldown combined with LC/MS/MS, we found that nonmuscle myosin IIA (MYH9) was a novel substrate of PTP1B in ESCC cells. PTP1B dephosphorylated MYH9 at Y1408, by which PTP1B up-regulated EGFR expression and enhanced cell migration and invasion in ESCC. In conclusion, our study first reported that PTP1B was the positive regulator of EGFR by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion, which revealed the regulatory mechanism of PTP1B-MYH9-EGFR axis in ESCC.

A novel MYH14 mutation in a Chinese family with autosomal dominant nonsyndromic hearing loss.

BACKGROUND: MYH14 gene mutations have been suggested to be associated with nonsyndromic/syndromic sensorineural hearing loss. It has been reported that mutations in MYH14 can result in autosomal dominant nonsyndromic deafness-4A (DFNA4). METHODS: In this study, we examined a four-generation Han Chinese family with nonsyndromic hearing loss. Targeted next-generation sequencing of deafness genes was employed to identify the pathogenic variant. Sanger sequencing and PCR-RFLP analysis were performed in affected members of this family and 200 normal controls to further confirm the mutation. RESULTS: Four members of this family were diagnosed as nonsyndromic bilateral sensorineural hearing loss with postlingual onset and progressive impairment. A novel missense variant, c.5417C > A (p.A1806D), in MYH14 in the tail domain of NMH II C was successfully identified as the pathogenic cause in three affected individuals. The family member II-5 was suggested to have noise-induced deafness. CONCLUSION: In this study, a novel missense mutation, c.5417C > A (p.A1806D), in MYH14 that led to postlingual nonsyndromic autosomal dominant SNHL were identified. The findings broadened the phenotype spectrum of MYH14 and highlighted the combined application of gene capture and Sanger sequencing is an efficient approach to screen pathogenic variants associated with genetic diseases.

Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI.

Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

Kinematics of the lever arm swing in myosin VI.

Myosin VI (MVI) is the only known member of the myosin superfamily that, upon dimerization, walks processively toward the pointed end of the actin filament. The leading head of the dimer directs the trailing head forward with a power stroke, a conformational change of the motor domain exaggerated by the lever arm. Using a unique coarse-grained model for the power stroke of a single MVI, we provide the molecular basis for its motility. We show that the power stroke occurs in two major steps. First, the motor domain attains the poststroke conformation without directing the lever arm forward; and second, the lever arm reaches the poststroke orientation by undergoing a rotational diffusion. From the analysis of the trajectories, we discover that the potential that directs the rotating lever arm toward the poststroke conformation is almost flat, implying that the lever arm rotation is mostly uncoupled from the motor domain. Because a backward load comparable to the largest interhead tension in a MVI dimer prevents the rotation of the lever arm, our model suggests that the leading-head lever arm of a MVI dimer is uncoupled, in accord with the inference drawn from polarized total internal reflection fluorescence (polTIRF) experiments. Without any adjustable parameter, our simulations lead to quantitative agreement with polTIRF experiments, which validates the structural insights. Finally, in addition to making testable predictions, we also discuss the implications of our model in explaining the broad step-size distribution of the MVI stepping pattern.