果蝇非常规肌球蛋白的结构与功能

肌球蛋白是一类重要的分子马达,可以将ATP水解产生的能量转化成动能,沿由肌动蛋白组成的细丝运动。肌球蛋白构成一个大的基因家族,在许多细胞活动中起着重要作用,包括肌肉收缩、胞内转运、听觉、视觉等。果蝇基因组有13种肌球蛋白基因,包括2种常规肌球蛋白和11种非常规肌球蛋白。本文综述了近年来果蝇非常规肌球蛋白的研究进展。     

A unique ATP hydrolysis mechanism of single-headed processive myosin, myosin IX

Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.     

Calcium regulates scallop muscle by changing myosin flexibility

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called “thick filament regulation” is quite different to vertebrate striated muscle which is switched on and off via “thin filament regulation” whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation. This paper is not being submitted elsewhere and the authors have no competing financial interests     

Kinetic mechanism of myosin IXB and the contributions of two class IX-specific regions

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.     

Discovery of myosin I and Pollard-san

In this short review, I describe a brief history of the discovery of myosin I isolated from Acanthamoeba in 1973 by Tom Pollard and Ed Korn. Today, myosins form a large “family tree” that includes more than 30 types of myosins. I discuss the importance of the relationship among actin, myosin, and other actin-binding proteins, many of which were pioneered by Pollard-san (“-san” is a Japanese honorific suffix showing respect, politeness and friendship). At the first conference devoted to actin, Pollard-san, Korn-san, and I discussed the importance of the nucleotide bound at the two ends of the actin filament. I conclude that life is a dynamic accumulation of molecule-molecule bindings, and although we do not yet know how they coordinate with each other to operate a living cell, many enthusiastic and excellent researchers like Pollard-san will unveil mechanisms that will show us what life really looks like.     

Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament

Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the “Driven by Detachment (DbD)” mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.     

Kinetic mechanism of non-muscle myosin IIB: functional adaptations for tension generation and maintenance

Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.     

Isolation of myosin XI genes from the Closterium peracerosum-strigosum-littorale complex and analysis of their expression during sexual reproduction

Myosins comprise a large superfamily of molecular motors that generate mechanical force in ATP-dependent interactions with actin filaments. On the basis of their conserved motor domain sequences, myosins can be divided into at least 17 classes, 3 of which (VIII, XI, XIII) are found in plants. Although full sequences of myosins are available from several species of green plants, little is known about the functions of these proteins. Additionally, sequence information for algal myosin is incomplete, and little attention has been given to the molecular evolution of myosin from green plants. In the present study, the Closterium peracerosum-strigosum-littorale complex was used as a model system for investigating a unicellular basal charophycean alga. This organism has been well studied with respect to sexual reproduction between its two mating types. Three types of partial sequences belonging to class XI myosins were obtained using degenerate primers designed to amplify motor domain sequences. Real-time polymerase chain reaction analysis of the respective myosin genes during various stages of the algal life cycle showed that one of the genes was more highly expressed during sexual reproduction, and that expression was cell-cycle-dependent in vegetatively grown cells.     

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Kinetic analysis of the slow skeletal myosin MHC-1 isoform from bovine masseter muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.     

Functional adaptation between yeast actin and its cognate myosin motors

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.     

Effect of trinitrophenylation on myosin ATPase

1. The enzymic and actin binding properties of myosins trinitrophenylated to different extents in the presence or absence of ATP have been studied.

2. The enzymic properties of myosin trinitrophenylated in the absence of ATP are different from those of myosin treated in the presence of ATP even on trinitrophenylating an equal number of lysyl residues. On trinitrophenylation in the absence of ATP the EDTA-(K⁺-)activated ATPase and Ca²⁺-activated ATPase decrease while the Mg²⁺-activated ATPase considerably increases. In the presence of ATP the enzymic properties of myosin are much less affected by trinitrophenylation.

3. The actin binding capacity of trinitrophenylated myosin does not change, although its enzymic properties may be greatly altered, and even if its property to be activated by actin is completely lost.     

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.     

An electromechanical model of myosin molecular motors

There is a long-running debate on the working mechanism of myosin molecular motors, which, by interacting with actin filaments, convert the chemical energy of ATP into a variety of mechanical work. After the development of technologies for observing and manipulating individual working molecules, experimental results negating the widely accepted 'lever-arm hypothesis' have been reported. In this paper, based on the experimental results so far accumulated, an alternative hypothesis is proposed, in which motor molecules are modelled as electromechanical components that interact with each other through electrostatic force. Electrostatic attractive force between myosin and actin is assumed to cause a conformational change in the myosin head during the attachment process. An elastic energy resulting from the conformational change then produces the power stroke. The energy released at the ATP hydrolysis is mainly used to detach the myosin head from actin filaments. The mechanism presented in this paper is compatible with the experimental results contradictory to the previous theories. It also explains the behavior of myosins V and VI, which are engaged in cellular transport and move processively along actin filaments.     

The structural basis for the large powerstroke of myosin VI

Due to a unique addition to the lever arm-positioning region (converter), class VI myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized myosin classes. However, the large size of the myosin VI lever arm swing (powerstroke) cannot be explained by our current view of the structural transitions that occur within the myosin motor. We have solved the crystal structure of a fragment of the myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it provides a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed myosin classes.     

Effect of actin filament length and filament number concentration on the actin-activated ATPase activity of Acanthamoeba myosin I

The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+-ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F-actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I.     

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.     

Actin and myosin in Gregarina polymorpha

Actin and two class XIV unconventional myosins have been cloned from Gregarina polymorpha, a large protozoan parasite inhabiting the gut of the mealworm Tenebrio molitor. These proteins were most similar to their homologues expressed in the coccidian and haemosporidian Apicomplexa such as Toxoplasma and Plasmodium despite the significant morphological differences among these parasites. Both actin and G. polymorpha myosin A (GpMyoA), a 92.6-kDa protein characterized by a canonical myosin head domain and short, highly basic tail, localized to both the longitudinally-disposed surface membrane folds (epicytic folds) of the parasite as well as to the subjacent rib-like myonemes that gird the parasite cortex. G. polymorpha myosin B (GpMyoB), a 96.3-kDa myosin, localized exclusively to the epicytic folds of the parasite. Both myosins were tightly associated with the cortical cytoskeleton and were solubilized only with a combination of high salt and detergent. Both GpMyoA and GpMyoB could bind to actin in an ATP-sensitive fashion. The distribution of actin and the unconventional myosins in G. polymorpha was consistent with their proposed participation in both the rapid (1-10 microm/sec) gliding motility exhibited by the gregarines as well as the myoneme-mediated bending motions that have been observed in these parasites.     

Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.     

The converter domain modulates kinetic properties of Drosophila myosin

Recently the converter domain, anintegral part of the "mechanical element" common to all molecularmotors, was proposed to modulate the kinetic properties ofDrosophila chimeric myosin isoforms. Here we investigatedthe molecular basis of actin filament velocity(V_actin) changes previously observed with thechimeric EMB-IC and IFI-EC myosin proteins [the embryonic body wallmuscle (EMB) and indirect flight muscle isoforms (IFI) with geneticsubstitution of the IFI and EMB converter domains, respectively]. Inthe laser trap assay the IFI and IFI-EC myosins generate the sameunitary step displacement (IFI = 7.3 ± 1.0 nm, IFI-EC = 5.8 ± 0.9 nm; means ± SE). Thus converter-mediateddifferences in the kinetics of strong actin-myosin binding, rather thanthe mechanical capabilities of the protein, must account for theobserved V_actin values. Basal andactin-activated ATPase assays and skinned fiber mechanical experimentsdefinitively support a role for the converter domain in modulating thekinetic properties of the myosin protein. We propose that the converterdomain kinetically couples the P_i and ADP release stepsthat occur during the cross-bridge cycle.