Effect of H-protein on the formation of myosin filaments and light meromyosin paracrystals

H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.     

Inhibition of smooth muscle myosin's activity and assembly by an anti-rod monoclonal antibody.

Monoclonal antibodies specific for the rod region can affect smooth muscle myosin's motor properties. Actin movement by phosphorylated myosin was inhibited by an antibody (LMM.4) which binds to the COOH-terminal end of the coiled-coil rod, a region thought to be involved in filament assembly. The actin-activated ATPase activity of the myosin-antibody LMM.4 complex was also reduced 10-fold at actin concentrations that gave maximal turnover rates with filamentous myosin. Metal-shadowing of the phosphorylated myosin-antibody complex at low ionic strength showed small bundles of parallel extended molecules, instead of filaments. Five other anti-rod antibodies had little or no effect on myosin's ability to act as a motor. This is the first demonstration that a muscle myosin's activity is affected by its state of assembly. A common theme that emerges from the studies on both muscle and non-muscle myosins is that assembly into a filamentous structure stimulates the activity of the individual myosin molecules.     

Interaction between vertebrate skeletal and uterine muscle myosins and light meromyosins

The specific contributions of this work may be summarized as follows: (a) No hybridization of uterine and skeletal myosin occurs at pH 6.0 although previous studies have shown that hybridization does occur at pH 6.5 (B. Kaminer et al. 1976. J. Mol. Biol. 100:379-386) or 7.0 (T. Pollard. 1975. J. Cell Biol. 67:93-104) (b) Hybridization of uterine and skeletal light meromyosins (LMM) occurs at pH 7.0 but not at pH 6.0, which is analogous to the hybridization of myosins. (c) In hybridized paracrystals there is a uniform distribution of both uterine and skeletal LMM molecules because all the paracrystals have only one axial repeat pattern. This makes it highly likely that in hybridized filaments the two myosins are also uniformly distributed throughout the filaments. (d) The 14-nm repeat of white bands observed in paracrystals of uterine LMM formed at pH 6.0, compared with the 14-nm repeat of dark bands observed with skeletal LMM under the same conditions, probably reflects differences in surface charge density along the different LMM molecules.     

Role of magnesium binding to myosin in controlling the state of cross-bridges in skeletal rabbit muscle

The effect of Mg2+ on the disposition of myosin cross-bridges was studied on myofibrils and synthetic myosin and rod filaments by employing chymotryptic digestion and chemical cross-linking methods. In the presence of low Mg2+ concentrations (0.1 mM), the proteolytic susceptibility at the heavy meromyosin/light meromyosin (HMM/LMM) junction in these three systems sharply increases over the pH range from 7.0 to 8.2. Such a change has been previously associated with the release of myosin cross-bridges from the filament surface [Ueno, H., & Harrington, W.F. (1981) J. Mol. Biol. 149, 619-640]. Millimolar concentrations of Mg2+ block or reverse this charge-dependent transition. Rod filaments show the same behavior as myosin filaments, indicating that the low-affinity binding sites for Mg2+ are located on the rod portion of myosin. The interpretation of these results in terms of Mg2+-mediated binding of cross-bridges to the filament backbone is supported by cross-linking experiments. The normalized rate of S-2 cross-linking in rod filaments at pH 8.0, kS-2/kLMM, increases upon addition of Mg2+ from 0.30 to 0.65 and approaches the cross-linking rate measured at pH 7.0 (0.75), when the cross-bridges are close to the filament surface. In rod filaments prepared from oxidized rod particles, chymotryptic digestion proceeds both at the S-2/LMM junction and at a new cleavage site located in the N-terminal portion of the molecule. Kinetic analysis of digestion rates at these two sites reveals that binding of Mg2+ to oxidized myosin rods has a similar effect at both sites over the pH range from 7.0 to 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

The purification and characterization of a globular subfragment of Acanthamoeba myosin II that is fully active when cross-linked to F-actin

Acanthamoeba myosin II contains two heavy chains of Mr 185,000 and two pairs of light chains of Mr 17,500 and 17,000. We now report the purification of a globular proteolytic 103-kDa subfragment of myosin II which contained a 68-kDa NH2-terminal segment of the heavy chain and one pair of intact light chains. The myosin II head fragment expressed full Ca2+-ATPase activity but its actin-activated Mg2+-ATPase activity had a Vmax of only 0.07 s-1 compared to 1.9 s-1 (per head) for filaments of native unphosphorylated myosin II. The head fragment had a similar KATPase to that of filaments (5 versus 4 microM) and about 75% of the head fraction could bind to F-actin in the presence of ATP with a Kbinding of 5.6 microM. The Kbinding of the head fragment may be similar to that of individual heads in the native myosin II filaments although the experimentally determined apparent Kbinding for filaments is much lower, 0.3 microM. The head fragment was covalently cross-linked to F-actin in the absence of nucleotide using the zero length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked actin-myosin head complex hydrolyzed MgATP at a rate equivalent to Vmax for the active dephosphorylated native myosin II. These data indicate that the isolated head fragment had intact catalytic and actin-binding domains but that it bound to F-actin in the presence of ATP in a relatively inactive conformation. When covalently cross-linked to F-actin the head fragment was apparently locked into a catalytically fully active conformation.     

Flexibility of Acanthamoeba myosin rod minifilaments.

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. II. Effect of modification of the Sa thiol group on superprecipitation and clearing.

The effect of Sa modification with NEM, which activates Mg2+-ATPase through an enhancement of the association of actin and myosin, was investigated on the superprecipitation, clearing and Mg2+-ITPase of myosin B with reference to the effect of S1-blocking. 1. Superprecipitation induced by ATP was markedly enhanced by Sa-blocking even at high concentrations of Mg2+ and substrate; this may be due to an increase in the affinity of myosin and actin on blocking Sa. 2. Nevertheless, neither ITP-induced superprecipitation nor Mg2+-ITPase was affected by Sa modification. 3. Blocking of S1 brought about the inhibition of ATP- and ITP-induced superprecipitation and Mg2+-ITPase activity, suggesting that S1-blocking decreases the affinity of myosin and actin. 4. Sa-blocked myosin B showed greater resistance to clearing by ATP, especially in the presence of Ca2+ ions, whereas in the clearing response of actomyosin gel to PPi no difference between Sa-blocked and unmodified myosins B was observed. On the other hand, the clearing response of myosin B became more sensitive to both ATP and PPi on blocking S1. Based on the above results and preliminary data suggesting that Sa is located in LMM, the interaction of myosin filaments and actin filaments under physiological conditions is discussed.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.     

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.     

Solubility-determining domain of smooth muscle myosin rod

Chymotryptic digestion of chicken gizzard light meromyosin (LMM) produced a 72 kDa core fragment, which was fully soluble at 150 mM KCl, pH 6.5–7.5. The fragment showed weak self-association at 50 mM KCl. The homology of the N-terminus amino acid sequence of this fragment with the sequence of the rabbit skeletal myosin rod suggested that the N-terminus of the core fragment originated 5 kDa from the hinge common to both smooth and skeletal myosin rod. Sedimentation experiments indicated that the domain specifying the insolubility of the intact LMM was 13 kDa long. Progressive proteolytic shortening of this region produced LMM fragments of progressively increasing solubility. Electron microscopy of segments formed from full-length LMM and from LMM core suggested that this 13 kDa domain specified the 43 nm parallel and antiparallel molecular overlaps characteristic of self-assembled intact myosin.     

Proteolytic substructure of brain myosin

Individual bovine brain myosin molecules visualized by electron microscopy consist of two globular heads and a fibrous tail, like myosin molecules from other sources. Brain myosin, however, showed much lower solubility at moderate to high ionic strength (0.2 to 0.4 M KCl) than gizzard myosin, and the filaments formed at low ionic strength in the presence of Mg2+ were fairly resistant to low concentrations of ATP, by which gizzard myosin filaments were completely solubilized. Brain myosin was digested with low concentrations of papain, alpha-chymotrypsin, or trypsin, and the fragmentation patterns were analyzed by means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation at low ionic strength, and electron microscopy of the fragments produced. The results indicate that all of the proteases cleave the myosin molecule primarily at sites located in the neck or in the head close to the neck, suggesting that the brain myosin molecule contains a hinge region or an open peptide stretch around these sites. The differences as well as the similarities between the proteolytic fragmentation patterns of brain myosin and other myosins are discussed.     

The axial repeats in paracrystals of light meromyosin and its complex with C-protein

We examined the axial repeats in electron micrographs of three types of negatively stained paracrystals (two tactoid- and one sheet-like type) of rabbit light meromyosin (LMM) and its complex with C-protein characterized previously by similar axial period of about 43.0 nm. Assuming for the axial repeat in type II tactoids the value of 42.93 +/- 0.05 nm as it was determined by X-ray diffraction technique (Yagi and Offer 1981), we found average axial repeats in type I tactoid and in sheet-like paracrystal of 42.93 +/- 0.75 nm and 43.50 +/- 0.62 nm respectively. Analyzing the micrographs where the two types paracrystals are located side-by-side we determined rather accurately the average ratio of axial repeat in sheet-like paracrystal to that in type I tactoid (1.013 +/- 0.002). Taking 42.93 nm as the axial repeat in type I tactoid, the axial repeat in sheet-like paracrystal was found to be 43.50 +/- 0.08 nm. C-protein binds to LMM with the period of the underlying LMM paracrystals and independently of the value of their axial repeats. Two different axial repeats (42.9 nm and 43.5 nm) revealed for LMM paracrystals in this study precisely coincide with the average repeat periods of myosin crossbridges along the thick filaments found for different physiological states of skeletal muscles (Lednev and Kornev 1987). Molecular basis for the appearance of two structural states in LMM paracrystals and in the shafts of thick filaments are discussed.     

Expression and characterization of human lamin C

We have expressed human lamin C cDNA in E. coli using a modification of the pLcII vector system. Protein produced in this way had seven additional amino acids at its N-terminus, but retained key lamin structural and assembly properties. The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system. Shadowed preparations of expressed lamin C showed the presence of 50-nm rod-like particles that closely resembled those observed for native material. Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains. The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained. We also used site-specific mutagenesis to engineer a lamin fragment that lacked the C-terminal non-helical domain of the molecule. This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C-terminal non-helical domain did not contain information vital for lamin assembly.     

Bending of smooth muscle myosin rod

Electron microscopy of mammalian smooth muscle myosin rods showed them to be 153 +/- 7 nm (SD) long, and to bend sharply (greater than 90 degrees) but infrequently, and pH independently (range 6.5-9.5), at a single site 45 +/- 4 nm from one end of the molecule. Light meromyosin (LMM) preparations were 99 +/- 10 nm long, and showed no bends. Intrinsic viscosity vs temperature plots for rods and LMM indicated that neither fragment changed in flexibility in the range 4-40 degrees C. Peptide mapping in the presence and absence of SDS established that the proteolytic susceptibility of the hinge at the N terminus of LMM reflects the presence of locally different structure, and not simply a clustering of susceptible residues. The isolated smooth muscle myosin rod thus contains only a single hinge, having significant stiffness, and lacks the second bend seen under certain conditions in the intact molecule.     

Determination of quantitative parameters of the binding of F-protein (phosphofructokinase) with myosin and localization of binding sites on the myosin molecule

To determine the localization of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 M KCl, 10 mM K-phosphate pH 6.5 was studied, using sedimentation, electron microscopic and optical diffraction methods. Sedimentation experiments showed that F-protein binds to myosin and myosin rod rather than to light meromyosin or S-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 X 10(5) M-1 and 2.1 X 10(5) M-1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of F-protein binding sites on myosin calculated per chain weight of 80 000 is 5 +/- 1. The sedimentation results were confirmed by electron microscopic data. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added before or after filamentogenesis. A comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones revealed a marked enhancement of meridional reflection at (14.3 nm)-1 in the latter case.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.     

Hydrolytically induced allosteric change in the heavy chain of intact myosin involving nonessential thiol groups.

The two globular head portions, each bearing an active site, contain an uncleaved heavy chain when isolated by chymotrypsin from intact myosin. By specific labeling with radioactive N-ethylmaleimide the essential thiol 1 and thiol 2 groups were found to reside in this heavy chain. In intact myosin nonessential thiol 3 groups become the most reactive during ATP hydrolysis above 15 degrees C. These thiol 3 groups are located in a portion of the myosin heavy chain which appears as a fragment with an apparent molecular weight of 11 000 during proteolysis. The facts that this fragment is produced in an almost 1: 1 molar ratio with the head heavy chain and that it bears unblocked N-terminal amino groups whereas the heavy chain does not and is not contained in the rod portion of the myosin molecule indicate that it may orginate from the heavy chains in the neck region where the heads are joined to the rod. Since this fragment is removed by ion-exchange chromatography, it is not part of the functioning head and hence not involved in the active site. As its nonessential thiol 3 groups are rendered the most reactive of all thiol groups in the enzyme-product complex M**ADP.Pi, the hydrolytic step induces an allosteric conformational change in the neck region of intact myosin.     

Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties.

The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.     

Actin and myosin-linked calcium regulation in the nematode Caenorhabditis elegans. Biochemical and structural properties of native filaments and purified proteins.

Calcium regulation of actomyosin activity in the nematode, Caenorhabditis elegans, has been studied with purified proteins and crude thin filaments. Actin and tropomyosin have been purified from C. elegans and shown to be similar in most respects to actin and tropomyosin from rabbit skeletal muscle. The actin comigrates with rabbit actin on polyacrylamide-sodium dodecyl sulfate gel electrophoresis, forms similar filaments and paracrystals, and activates the Mg2+-ATPase of rabbit myosin heads as efficiently as rabbit actin. Nematode tropomyosin has a greater apparent molecular weight (estimated by mobility on polyacrylamide-sodium dodecyl sulfate gels) than the rabbit protein, yet it forms Mg2+-paracrystals with a slightly shorter periodicity. Native thin filaments extracted from nematodes activate rabbit myosin subfragment 1 Mg2+-ATPase in a calcium sensitive manner; the extent of activation is threefold greater in 0.2 mM CaCl2 than in the absence of calcium. This observation suggests that the thin filaments contain components which are functionally equivalent to vertebrate troponins. Calcium is also required for maximal activation of the Mg2+-ATPase of purified nematode myosin by pure rabbit F-actin. C. elegans therefore has both myosin and thin filament-linked calcium regulatory systems. The origin of the actin, tropomyosin, and myosin from different tissues and the use of genetic analysis to answer questions about assembly and function in vivo are discussed.