Myosin Tail Research Articles

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β–β associations found in radixin–adhesion molecule complexes. We also revealed direct interactions between the MyTH4–FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.

Characterization of a Myosin VII MyTH/FERM Domain

A group of closely related myosins is characterized by the presence of at least one MyTH/FERM ( myosin tail homology; band 4.1, ezrin, radixin, moesin) domain in their C-terminal tails. This domain interacts with a variety of binding partners, and mutations in either the MyTH4 or the FERM domain of myosin VII and myosin XV result in deafness, highlighting the functional importance of each domain. The N-terminal MyTH/FERM region of Dictyostelium myosin VII (M7) has been isolated as a first step toward gaining insight into the function of this domain and its interaction with binding partners. The M7 MyTH4/FERM domain (MF1) binds to both actin and microtubules in vitro, with dissociation constants of 13.7 and 1.7 μM, respectively. Gel filtration and UV spectroscopy reveal that MF1 exists as a monomer in solution and forms a well-folded, compact conformation with a high degree of secondary structure. These results indicate that MF1 forms an integrated structural domain that serves to couple actin filaments and microtubules in specific regions of the cytoskeleton.

Protein multi-scale organization through graph partitioning and robustness analysis: application to the myosin–myosin light chain interaction

Despite the recognized importance of the multi-scale spatio-temporal organization of proteins, most computational tools can only access a limited spectrum of time and spatial scales, thereby ignoring the effects on protein behavior of the intricate coupling between the different scales. Starting from a physico-chemical atomistic network of interactions that encodes the structure of the protein, we introduce a methodology based on multi-scale graph partitioning that can uncover partitions and levels of organization of proteins that span the whole range of scales, revealing biological features occurring at different levels of organization and tracking their effect across scales. Additionally, we introduce a measure of robustness to quantify the relevance of the partitions through the generation of biochemically-motivated surrogate random graph models. We apply the method to four distinct conformations of myosin tail interacting protein, a protein from the molecular motor of the malaria parasite, and study properties that have been experimentally addressed such as the closing mechanism, the presence of conserved clusters, and the identification through computational mutational analysis of key residues for binding.

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β-β associations found in radixin-adhesion molecule complexes. We also revealed direct interactions between the MyTH4-FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.

Unusual N-glycan Structures Required for Trafficking Toxoplasma gondii GAP50 to the Inner Membrane Complex Regulate Host Cell Entry Through Parasite Motility

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.

Light‐Triggered Myosin Activation for Probing Dynamic Cellular Processes

Myosin II is an ATPase motor protein essential for many cellular functions including cell migration[1] and division.[2] In nonmuscle cells, myosin modulates protrusions at the leading edge and promotes retraction at the trailing edge during migration,[3] while during cytokinesis, myosin is required for contraction of the cleavage furrow.[4] For nonmuscle myosin, these varied functions are regulated by phosphorylation of the associated myosin regulatory light chain (mRLC) protein at Ser19, which activates the myosin complex to promote myosin assembly, contractility, and stress fiber formation.[5] Upon phosphorylation of the mRLC at both Thr18 and Ser19, these activities are further enhanced.[6 ] The dramatic effects of phosphorylation can also be recapitulated in vitro. Specifically, myosin and the proteolytic derivative heavy meromyosin (HMM),[7] which contains only one-third of the C-terminal myosin tail, exhibit low in vitro activities when associated with the nonphosphorylated mRLC. Phosphorylation of Ser19 amplifies actin-activated ATPase activities 10 – 1000-fold[8] and leads to myosin-mediated actin translocation.[9]

MyTH4, independent of its companion FERM domain, affects the organization of an intramacronuclear microtubule array and is involved in elongation of the macronucleus in Tetrahymena thermophila

Myo1 is a class XIV Tetrahymena myosin involved in amitotic elongation and constriction of the macronucleus into two subnuclei at cell division. Elongation of the macronucleus is accompanied by elongation of an intramacronuclear microtubule array, which is oriented parallel to the axis of nuclear elongation. Elongation of the macronucleus often fails to occur or is only partially completed in a MYO1 knockout, and division of the macronucleus is frequently uncoupled from cytokinesis. Myo1 contains a myosin tail homology 4 (MyTH4) and a band 4.1, ezrin, radixin, moesin homology (FERM) domain. Recently, we used green fluorescent protein (GFP) fusions to demonstrate that the entire FERM domain, independent of MyTH4, is essential for localization of FERM to the cytoskeleton and does not appear to directly affect nuclear division. Antiactin coprecipitates GFP-FERM, tubulin, actin, and Myo1. The immunoprecipitated GFP-FERM cosediments with either exogenous F-actin or exogenous microtubules. Here, we show that overexpressed GFP-MyTH4 colocalized with antitubulin to intramacronuclear microtubules. Ninety percent of overexpressing cells assembled intramacronuclear microtubules that did not become organized into a parallel array. Amitosis did not advance in the absence of the parallel array of intramacronuclear microtubules. Five percent of overexpressing cells organized the parallel array, but the microtubules and the macronucleus did not achieve full elongation. Partially elongated macronuclei constricted without cytokinesis. Antiactin coprecipitated GFP-MyTH4, tubulin, and actin. AntiGFP pulled down GFP-MyTH4, tubulin, and actin. GFP-MyTH4 cosedimented with either exogenous microtubules or exogenous F-actin. A novel finding from this study is that MyTH4 and FERM have overlapping and distinct roles in the function of a myosin.

Rapid discovery of inhibitors of Toxoplasma gondii using hybrid structure-based computational approach

Toxoplasma (T.) gondii, the causative agent of toxoplasmosis, is a ubiquitous opportunistic pathogen that infects individuals worldwide, and is a leading cause of severe congenital neurologic and ocular disease in humans. No vaccine to protect humans is available, and hypersensitivity and toxicity limit the use of the few available medicines. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. Using the Hybrid Structure Based (HSB) method, we have previously identified small molecule inhibitors of P. falciparum that seem to target a novel protein-protein interaction between the Myosin tail interacting protein and myosin light chain. This pathway has been hypothesized to be involved in invasion of host erythrocytes by the parasite and is broadly conserved among the apicomplexans. Guided by similar computational drug design approaches, we investigated this series of small molecules as potential inhibitors of T. gondii. Compound C3-21, identified as the most active inhibitor in this series, exhibited an IC(50) value ~500 nM against T. gondii. Among the 16 structural analogs of C3-21 tested thus far, nine additional compounds were identified with IC(50) values <10.0 μM. In vitro assays have revealed that C3-21 markedly limits intracellular growth of T. gondii tachyzoites, but has no effect on host cell human foreskin fibroblasts (HFF) at concentrations more than a log greater than the concentration that inhibits the parasites.

Structure of MyTH4-FERM Domains in Myosin VIIa Tail Bound to Cargo

The unconventional myosin VIIa (MYO7A) is one of the five proteins that form a network of complexes involved in formation of stereocilia. Defects in these proteins cause syndromic deaf-blindness in humans [Usher syndrome I (USH1)]. Many disease-causing mutations occur in myosin tail homology 4-protein 4.1, ezrin, radixin, moesin (MyTH4-FERM) domains in the myosin tail that binds to another USH1 protein, Sans. We report the crystal structure of MYO7A MyTH4-FERM domains in complex with the central domain (CEN) of Sans at 2.8 angstrom resolution. The MyTH4 and FERM domains form an integral structural and functional supramodule binding to two highly conserved segments (CEN1 and 2) of Sans. The MyTH4-FERM/CEN complex structure provides mechanistic explanations for known deafness-causing mutations in MYO7A MyTH4-FERM. The structure will also facilitate mechanistic and functional studies of MyTH4-FERM domains in other myosins.

Evidence for Pre- and Post-Power Stroke of Cross-Bridges of Contracting Skeletal Muscle

We examined orientational fluctuations of a few molecules of myosin in working ex-vivo skeletal myofibril. Light Chain 1 (LC1) of myosin was labeled with fluorescent dye and exchanged with native LC1 of skeletal myofibril. A small volume within the A-band (∼10−16L), containing on average 3-4 fluorescent myosin molecules, was observed by confocal microscopy. The myofibrils were cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent shortening. During muscle contraction myosin tail (containing LC1) undergoes cyclic fluctuations of orientation. We measured these fluctuations by recording the parallel and perpendicular components of fluorescent light emitted by myosin LC1-bound fluorophore. The histograms of fluctuations of fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms of contracting muscles could be fitted by at least two Gaussians. This provides evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power stroke.

Myo1 provides a ring in the tail

![Figure][1] Yeast expressing the Myo1 tail (right) still form a primary septum (PS), which promotes cytokinesis in the absence of Myo1 motor activity. Fang et al. describe two distinct pathways that target the tail of yeast myosin II to the bud neck to direct cell division. Myosin II

Simultaneous Observation of Tail and Head Movements of Myosin V during Processive Motion

Processive stepping of myosin Va (myoV) has been tracked by monitoring either the tail position (center of mass) or the position of one or both heads. Here, we combine these two approaches by attaching a quantum dot to one of the motor domains and a bead to the tail. Using laser trapping and total internal reflection microscopy, the position of one head and the tail are observed simultaneously as myoV moves processively on an actin filament bundle against the resistive load of the laser trap. The head moves one step (73 ± 10 nm) for every two steps of the tail (35 ± 9 nm). One tail step occurs concurrently with quantum dot-labeled head movement, whereas the other occurs with movement of the unlabeled head, consistent with a hand-over-hand model. Load increases the probability of the motor taking a back step. The back step is triggered by the motor taking a shorter forward step (head step, 68 ± 11 nm; tail step, 32 ± 10 nm), likely one actin monomer short of its preferred binding site. During a back step, the motor reverses its hand-over-hand motion, with the leading head detaching and reattaching to one of multiple actin sites behind the trailing head. After a back step, the motor can correct its mistake and step processively forward at resistive loads <0.7 piconewton or stall or detach at higher loads. Back stepping may provide a mechanism to ensure efficient cargo delivery even when myoV encounters obstacles within the actin cytoskeletal meshwork or when other motors are attached to the same cargo.

Structure-based Design of Novel Small-Molecule Inhibitors of Plasmodium falciparum

Malaria is endemic in most developing countries, with nearly 500 million cases estimated to occur each year. The need to design a new generation of antimalarial drugs that can combat the most drug-resistant forms of the malarial parasite is well recognized. In this study, we wanted to develop inhibitors of key proteins that form the invasion machinery of the malarial parasite. A critical feature of host-cell invasion by apicomplexan parasites is the interaction between the carboxy terminal tail of myosin A (MyoA) and the myosin tail interacting protein (MTIP). Using the cocrystal structure of the Plasmodium knowlesi MTIP and the MyoA tail peptide as input to the hybrid structure-based virtual screening approach, we identified a series of small molecules as having the potential to inhibit MTIP-MyoA interactions. Of the initial 15 compounds tested, a pyrazole-urea compound inhibited P. falciparum growth with an EC(50) value of 145 nM. We screened an additional 51 compounds belonging to the same chemical class and identified 8 compounds with EC(50) values less than 400 nM. Interestingly, the compounds appeared to act at several stages of the parasite's life cycle to block growth and development. The pyrazole-urea compounds identified in this study could be effective antimalarial agents because they competitively inhibit a key protein-protein interaction between MTIP and MyoA responsible for the gliding motility and the invasive features of the malarial parasite.

An unconventional myosin required for cell polarization and chemotaxis.

MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.

Myosin diversity in the diatom Phaeodactylum tricornutum

This report describes the domain architecture of ten myosins cloned from the pennate diatom Phaeodactylum tricornutum. Several of the P. tricornutum myosins show similarity to myosins from the centric diatom Thalassiosira pseudonana as well as to one myosin from the oomycete Phytophthora ramorum. The P. tricornutum myosins, ranging in size from 126 kDa to over 250 kDa, all possess the canonical head, neck and tail domains common to most myosins, though variations in each of these domains is evident. Among the features distinguishing several of the diatom myosin head domains are N-terminal SH3-like domains, variations in or near the P-loop and Loop 1 regions close to the nucleotide binding pocket, and extended converter domains. Variations in the length of the neck domain or lever arm, defined by the light chain-binding IQ motifs, are apparent with the different diatom myosins predicted to contain from one to nine IQ motifs. Protein domains found within the P. tricornutum myosin tails include regions of coiled-coil structure, ankyrin repeats, CBS domain pairs, a PB1 domain, a kinase domain and a FYVE-finger motif. As many of these features have never before been characterized in myosins of any type, it is likely that these new diatom myosins will expand the repertoire of known myosin behaviors.

Simultaneous Observation of Tail and Head Movements of Myosin V During Processive Motion Provides Insight into Its Stepping Dynamics

Processive stepping of myosin V (myoV) on actin has been studied either by tracking the position of the tail, which follows the motion of the molecule as a whole, or by tracking the position of one or both heads. Here we combine these two approaches, and attach a quantum dot (Qdot) to one of the motor domains, and a bead to the tail. Using optical trapping and total internal reflection microscopy, the position of one head and the tail are simultaneously observed as myoV moves processively on an actin filament against increasing load. Our results show that the head (Qdot) moves continually with 72.9±10.3 nm step size, while the tail (bead) moves with a step size of 34.7±8.6 nm. For every two tail steps, the head moves only one step. One of the tail steps takes place concurrently with the head step. Back steps were occasionally observed. Analysis shows that before taking a back step, the head moves 68±11nm while the tail moves 31.9±9.7nm, which suggests that the leading head lands on the 11th actin subunit instead of its normal 13th actin subunit. Interestingly, during a backstep the tail moves −28.6±13.7nm, while the step size distribution for the head shows multiple peaks. This suggests that the head has multiple binding positions along the actin filaments, while the tail has a more defined conformation. Our observation supports a hand-over-hand model for processive movement of myoV, and reveals the cause of the back stepping behavior of myosin V under physiologically relevant loading forces (<2pN).

Interaction and dynamics of the Plasmodium falciparum MTIP–MyoA complex, a key component of the invasion motor in the malaria parasite

The myosin tail domain interacting protein-myosin A (MTIP-MyoA) protein complex is an essential element of the motor driving invasion of red blood cells by the Plasmodium species that cause malaria. Here we report the key determinants of binding at the MTIP/MyoA interface, and the first structural study on the complex in solution using protein NMR.

Identification of Restricting Factors That Inhibit Swelling of Oxidized Myofibrils during Brine Irrigation

Porcine longissimus myofibrils were exposed to a hydroxyl radical-oxidizing system (10 microM FeCl3, 100 microM ascorbic acid, 1 mM H2O2) at pH 6.2 for 1-12 h. Chemical analyses (sulfhydryls, disulfide bonds, carbonyls) indicated mild protein oxidation along with almost 40% loss of protein extractability in low-ionic-strength brines (<or=0.4 M NaCl, 10 mM pyrophosphate, pH 6.2). Upon graded brine irrigation (0.2-->0.6 M NaCl) with pyrophosphate, the swelling of myofibrils and the dissolution of the A-band of oxidized myofibrils were less pronounced than those of nonoxidized. This restriction of myofibril swelling, caused largely by disulfide cross-linkages formed between oxidized myosin tails, was positioned on the transversely expansible thick filaments, reflecting a significant role and susceptibility of intra- as well as intermyofilamental structures.

A Myosin XI Tail Domain Homologous to the Yeast Myosin Vacuole-Binding Domain Interacts with Plastids and Stromules in Nicotiana benthamiana

The actin cytoskeleton plays a role in mobility of many different organelles in plant cells, including chloroplasts, mitochondria, Golgi, and peroxisomes. While progress has been made in identifying the myosin motors involved in trafficking of various plant organelles, not all of the cargoes mobilized by different members of the myosin XI family have yet been identified. The involvement of myosins in chloroplast positioning and mitochondrial movement was demonstrated by expression of a virus-induced gene silencing (VIGS) construct in tobacco. When VIGS with two different conserved sequences from a myosin XI motor was performed in plants with either GFP-labeled plastids or mitochondria, chloroplast positioning in the dark was abnormal, and mitochondrial movement ceased. Because these and prior observations have implicated a role for myosins and the actin cytoskeleton in plastid and stromule movement, we searched for myosin tail domains that could associate with plastids and stromules. While a yellow fluorescent protein (YFP) fusion with the entire tail region of myosin XI-F was usually found only in the cytoplasm, we observed that an Arabidopsis or Nicotiana benthamiana YFP::myosin XI-F tail domain homologous to the yeast myo2p vacuole-binding domain associated with plastids and stromules after transient expression in N. benthamiana. Taken together, these observations implicate myosin motor proteins in dynamics of plastids and stromules.

G.P.7.10 Investigation of the patho-biology of MYH7 myopathy mutations

The β-cardiac myosin (β-MyHC) protein is a molecular motor fundamental to both the contractile and structural properties of the muscle sarcomere. Mutations in the gene encoding β-MyHC (MYH7) cause multiple disease phenotypes: early-onset distal myopathy (MPD1), myosin storage myopathy (MSM), hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Mutations causing HCM and DCM are spread across almost the entire gene, while those causing MPD1 and MSM are confined to exons encoding the C-terminal light-meromyosin (LMM) region of β-MyHC. How mutations located in the same region of MYH7 cause such a wide phenotypic range is as yet unknown. To investigate structural and functional effects of different β-MyHC mutants, full-length MYH7 and rod/LMM domain regions of interest were cloned for expression in mammalian cells or for recombinant protein expression. Fusion to enhanced green fluorescent protein (EGFP) allowed visualisation of the location of wildtype (WT) or mutant β-cardiac myosin in C2C12 myoblast and myotube cultures and in COS7 cells. Both WT and mutant proteins were able to arrange into ordered, striated structures in differentiated C2C12 myotubes. Analysis in COS7 cells however, suggested mutant β-MyHC showed reduced ability to form higher order structures compared to the WT protein. Secondary-structure analysis of recombinant expressed myosin tail fragments by circular dichroism (CD) revealed that both WT and mutant proteins are almost entirely α-helical. Mutant myosin tails however, tend to show a slight reduction in α-helical content. This reduction is amplified when temperature is increased to replicate physiological conditions. CD melt curve analysis indicated mutant β-MyHC proteins have decreased thermostability. Overall, the effects of the MYH7 LMM mutations on measured properties are less marked than might have been expected from missense mutations to proline or deletion or insertion of an amino-acid in a coiled coil.