Ca2+ activates actin-filament sliding on scallop myosin but inhibits that on Physarum myosin

The actin-activated ATPase activity of Physarum myosin has been shown to be inhibited by microM levels of Ca2+, the mode of which is in contrast to the activating effect of Ca2+ on scallop myosin (Kohama, K. (1987) Adv. Biophys. 23, 149-182 for a review). To determine if Ca2+ regulates ATP-dependent sliding between actin and the myosins, fluorescent actin-filaments were allowed to move on the myosins fixed to a glass surface. The movement on Physarum and scallop myosins was inhibited and activated, respectively, by Ca2+. For this myosin-linked regulation to occur for Physarum myosin, myosin phosphorylation was shown to be a prerequisite.     

An unstable head-rod junction may promote folding into the compact off-state conformation of regulated myosins

The N-terminal region of myosin's rod-like subfragment 2 (S2) joins the two heads of this dimeric molecule and is key to its function. Previously, a crystal structure of this predominantly coiled-coil region was determined for a short fragment (51 residues plus a leucine zipper) of the scallop striated muscle myosin isoform. In that study, the N-terminal 10-14 residues were found to be disordered. We have now determined the structure of the same scallop peptide in three additional crystal environments. In each of two of these structures, improved order has allowed visualization of the entire N-terminus in one chain of the dimeric peptide. We have also compared the melting temperatures of this scallop S2 peptide with those of analogous peptides from three other isoforms. Taken together, these experiments, along with examination of sequences, point to a diminished stability of the N-terminal region of S2 in regulated myosins, compared with those myosins whose regulation is thin filament linked. It seems plain that this isoform-specific instability promotes the off-state conformation of the heads in regulated myosins. We also discuss how myosin isoforms with varied thermal stabilities share the basic capacity to transmit force efficiently in order to produce contraction in their on states.     

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.     

Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head

We have extended the X-ray structure determination of the complete scallop myosin head in the pre-power stroke state to 2.6 A resolution, allowing an atomic comparison of the three major (weak actin binding) states of various myosins. We can now account for conformational differences observed in crystal structures in the so-called "pliant region" at the motor domain-lever arm junction between scallop and vertebrate smooth muscle myosins. A hinge, which may contribute to the compliance of the myosin crossbridge, has also been identified for the first time within the regulatory light-chain domain of the lever arm. Analysis of temperature factors of key joints of the motor domain, especially the SH1 helix, provides crystallographic evidence for the existence of the "internally uncoupled" state in diverse isoforms. The agreement between structural and solution studies reinforces the view that the unwinding of the SH1 helix is a part of the cross-bridge cycle in many myosins.     

Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease

Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.     

Regulatory and essential light-chain interactions in scallop myosin. I. Protection of essential light-chain thiol groups by regulatory light-chains

The reactivity of the thiol groups of the essential light-chains of scallop myosin is greatly reduced by the presence of regulatory light-chains on myosin. The thiol groups of the essential light-chains react with iodoacetate only if the regulatory light-chains have been removed by treatment with EDTA. No alkylation of the essential light-chains could be detected in myosins containing regulatory light-chains (untreated or reconstituted myosins) after an overnight incubation with excess iodoacetate at 4 °C. In contrast, similar treatment alkylated two to three thiol groups of essential light-chains in desensitized myosins from which the regulatory light-chains had been removed. In addition, up to seven of the 20 heavy-chain thiols were also alkylated; however, the reactivity of the heavy-chain thiols did not depend on the presence of the regulatory light-chains. ATPase activities were not inhibited by alkylation with iodoacetate. Regulatory light-chains also protected essential light-chain thiols against reaction with N-iodoacetyl-N^′-(l-sulfo-5-naphthyl) ethylenediamine and against dansylation at pH 6.7, although treatment with these reagents caused a considerable loss of ATPase activities. Rebinding of the regulatory light-chains was impaired by alkylation. The results indicate an extensive interaction between the regulatory and the essential light-chains in scallop myosin.     

Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosins using electron paramagnetic resonance spectroscopy.

The binding of Ca²⁺, Mg²⁺ and Mn²⁺ to myosins from rabbit skeletal muscle, scallop striated adductor muscle and clam adductor muscle has been investigated. All three myosins bind two moles of divalent metal ion non-specifically and with high affinity (Mn²⁺ > Ca²⁺ > Mg²⁺). In addition, the molluscan myosins bind about a further two moles of Ca²⁺ specifically. Although rabbit myosin binds some Ca²⁺ in the presence of an excess of free Mg²⁺, this binding occurs at the nonspecific sites and should not be taken as evidence for a myosin-linked regulatory system of the type found in molluscan muscles. If such a system exists in vertebrate skeletal muscle, the homologous Ca²⁺-specific sites must be lost during the early stages of the myosin preparation.The characteristic electron paramagnetic resonance spectrum of the bound Mn²⁺ was utilized to confirm the homology of the non-specific sites in vertebrate and molluscan myosins. The sites are located on the “regulatory” class of light chain. Mn²⁺ bound to scallop myosin has a broad electron paramagnetic resonance spectrum, in contrast to the well-resolved spectra that it gives when bound to many other myosin species. This situation was exploited to identify homologous nonspecific, divalent metal-ion sites on the regulatory light chains from a variety of muscle types, including frog skeletal, rabbit cardiac, chicken gizzard and molluscan adductor muscles. When these light chains are combined with desensitized scallop myofibrils the electron paramagnetic resonance spectra of Mn²⁺ bound to the resultant hybrids are dominated by the signal from the non-specific site of the foreign regulatory light chain.     

Identification of a novel unconventional myosin from scallop mantle tissue.

We isolated a cDNA encoding a novel unconventional myosin from scallop mantle tissue (scallop unconventional myosin: ScunM) and determined the nucleotide sequence. It comprises 2,739 bp with 5' and 3'-noncoding sequences and has an open reading frame of 2,334 bp that encodes 778 amino acids. While ScunM has a motor domain and a short tail domain without having light chain-binding IQ motifs like myosin XIV, the deduced amino acid sequence exhibits low homology, 30-36%, to known myosins. Phylogenetic analysis of the motor domain suggested that ScunM belongs to a novel unconventional myosin class. ScunM has an insertion of 67 amino acids in the putative actin-binding site (loop2 site). Western blot analysis with an antibody produced against the N-terminal region revealed that ScunM was strongly expressed in the mantle and mantle pallial cell layer of scallop.     

Regulatory light chains in myosins.

Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity.Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA.There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.     

Isolation of a cDNA encoding the motor domain of nonmuscle myosin which is specifically expressed in the mantle pallial cell layer of scallop (Patinopecten yessoensis)

It has been reported that catch and striated muscle myosin heavy chains of scallop are generated through alternative splicing from a single gene [Nyitray et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12686-12690]. They suggested that the catch muscle type myosin was expressed in various tissues of scallop, including the gonad, heart, foot, and mantle. However, there have been no reports of the primary structure of myosin from tissues other than the adductor muscles. In this study, we isolated a cDNA encoding the motor domain of myosin from the mantle tissue of scallop (Patinopecten yessoensis), and determined its nucleotide sequence. Sequence analysis revealed that mantle myosin exhibited 65% identity with Drosophila non muscle myosin, 60% with chicken gizzard smooth muscle myosin, and 44% with scallop striated muscle myosin. The mantle myosin has inserted sequences in the 27 kDa domain of the head region, and has a longer loop 1 structure than those of scallop striated and catch muscle myosins. Phylogenetic analysis suggested that the mantle myosin is classified as a smooth/nonmuscle type myosin. Western blot analysis with antibodies produced against the N-terminal region of the mantle myosin revealed that this myosin was specifically expressed in the mantle pallial cell layer consisting of nonmuscle cells. Our results show that mantle myosin is classified as a nonmuscle type myosin in scallop.     

Akazara scallop myosins hybridized with DTNB-light chains of skeletal myosin and with regulatory light chains of gizzard myosin

Two different hybrid myosins were obtained by combining "desensitized" myosin (DM) of Akazara scallop striated adductor with rabbit skeletal DTNB-light chains (DTNB-LC) and with chicken gizzard regulatory light chains (GR-LC). Using the two hybrid myosins, the following were found: (a) DTNB-LC has an inhibitory effect on the Mg-ATPase activities of Akazara DM and acto-DM both in the absence of calcium and in its presence. (b) DTNB-LC also has an enhancing effect on the superprecipitation activity of acto-DM. (c) The Mg-ATPase activities of DM and acto-DM are made sensitive to calcium by GR-LC, regardless of whether GR-LC is phosphorylated or unphosphorylated. (d) However, the Mg-ATPase activity of acto-myosin hybridized with phosphorylated GR-LC is definitely higher than that of acto-myosin hybridized with unphosphorylated GR-LC.     

Photochemical mapping of the active site of myosin.

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the gamma-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.     

Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation

We have determined the primary structure of the myosin heavy chain (MHC) of the striated adductor muscle of the scallop Aequipecten irradians by cloning and sequencing its cDNA. It is the first heavy chain sequence obtained in a directly Ca(2+)-regulated myosin. The 1938-amino acid sequence has an overall structure similar to other MHCs. The subfragment-1 region of the scallop MHC has a 59-62% sequence identity with sarcomeric and a 52-53% identity with nonsarcomeric (smooth and metazoan nonmuscle) MHCs. The heavy chain component of the regulatory domain (Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'Neall-Hennessey, E., Melandri, F. D., and Szent-Gy?rgyi, A. G. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4771-4775) starts at either Leu-755 or Val-760. Ca(2+)-sensitive Trp residues (Wells, C., Warriner, K. E., and Bagshaw, C. R. (1985) Biochem. J. 231, 31-38) are located near the C-terminal end of this segment (residues 818-827). More detailed sequence comparison with other MHCs reveals that the 50-kDa domain and the N-terminal two-thirds of the 20-kDa domain differ substantially between sarcomeric and nonsarcomeric myosins. In contrast, in the light chain binding region of the regulatory domain (residues 784-844) the scallop sequence shows greater homology with regulated myosins (smooth muscle, nonmuscle, and invertebrate striated muscles) than with unregulated ones (vertebrate skeletal and heart muscles). The N-terminal 25-kDa domain also contains several residues which are preserved only in regulated myosins. These results indicate that certain heavy chain sites might be critical for regulation. The rod has features typical of sarcomeric myosins. It is 52-60% and 30-33% homologous with sarcomeric and nonsarcomeric MHCs, respectively. A Ser-rich tailpiece (residues 1918-1938) is apparently nonhelical.     

Amino acid sequences of the two kinds of regulatory light chains of adductor smooth muscle myosin from Patinopecten yessoensis

Smooth muscle myosin from scallop (Patinopecten yessoensis) adductor muscle contains two kinds of regulatory light chains (regulatory light chains a and b), and myosin having regulatory light chain a is suggested to be suitable for inducing "catch contraction" rather than myosin having regulatory light chain b (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673-681). The amino acid sequences of these two light chains were determined and compared. Regulatory light chain a consists of 161 amino acid residues, while regulatory light chain b consist of 156 amino acid residues. Amino acid substitutions and insertions were found only in the N-terminal regions of the sequences. The structural difference between the two light chains may contribute to the functional difference between myosins having regulatory light chains a and b.     

Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin

Three-dimensional reconstructions of “barbed” and “blunted” arrowheads (Craig et al., 1980) show that these two forms arise from arrangement of scallop myosin subfragments (S1) that appear about 40 Å longer in the presence of the regulatory light chain than in its absence. A similar difference in apparent length is indicated by images of single myosin subfragments in partially decorated filaments. The extra mass is located at the end of the subfragment furthest from actin, and probably comprises part of the regulatory light chain as well as a segment of the myosin heavy chain. The fact that barbed arrowheads are also formed by myosin subfragments from vertebrate striated and smooth muscles implies that the homologous light chains in these myosins have locations similar to that of the scallop light chain.The scallop light chain probably does not extend into the actin-binding site on the myosin head, and is therefore unlikely to interfere physically with binding. Rather, regulation of actin-myosin interaction by light chains may involve Ca²⁺-dependent changes in the structure of a region near the head-tail junction of myosin.The reconstructions suggest locations for actin and tropomyosin relative to myosin that are similar to those proposed by Taylor & Amos (1981) and are consistent with a revised steric blocking model for regulation by tropomyosin. The identification of actin from these reconstructions is supported by images of partially decorated filaments that display the polarity of the actin helix relative to that of bound myosin subfragments.     

LC2 involvement in the assembly of skeletal myosin filaments

The assembly of LC2-deficient myosin was studied under conditions where control and LC2-reassociated myosin assemble around the native length of about 1.5 microns. The aim of this work was to determine how loss of LC2 affects the assembly characteristics. The findings of this study can be summarized as follows: (a) LC2-deficient myosin assembles into two populations of filaments, one around 0.5 micron in length and the other around 1 micron in length. This suggests that loss of the LC2 perturbs the length-determining mechanism. (b) The population of filaments around 0.5 micron has a diameter around 14 nm and that around 1 micron a diameter around 22 nm. Neither diameter corresponds to the 18 nm obtained with the control and LC2-reassociated myosins, suggesting that the presence of LC2 may have a role in regulating the side-to-side assembly of the myosin rods. (c) Filaments assembled from LC2-deficient myosin tend to aggregate side-by-side, but not those assembled from control and LC2-reassociated myosin. (d) The presence of MgATP has no effect on the length distribution of LC2-deficient myosin filaments in contrast to the sharpening of the distribution observed with control and reassociated myosin.     

The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.     

PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN

The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60–95%) and chick pectoralis muscle (31–83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50–60%, identities), and some also matched unconventional myosins (33–50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles.     

Conformation and dynamics of the SH1-SH2 helix in scallop myosin

Atomic structures of scallop myosin subfragment 1(S1) with the bound MgADP, MgAMPPNP, and MgADP.BeF(x) provide crystallographic evidence for a destabilization of the helix containing reactive thiols SH1 (Cys703) and SH2 (Cys693). A destabilization of this helix was not observed in previous structures of S1 (from chicken skeletal, Dictyostelium discoideum, and smooth muscle myosins), including complexes for which solution experiments indicated such a destabilization. In this study, the factors that influence the SH1-SH2 helix in scallop S1 were examined using monofunctional and bifunctional thiol reagents. The rate of monofunctional labeling of scallop S1 was increased in the presence of MgADP and MgATPgammaS but was inhibited by MgADP.V(i) and actin. The resulting changes in ATPase activities of S1 were symptomatic of SH2 and not SH1 modification, which was confirmed by mass spectrometry analysis. With bifunctional reagents of various lengths, cross-linking did not occur on a short time scale in the absence of nucleotides. In the presence of MgADP, cross-linking was greatly enhanced for all of the reagents. These reactions, as well as the formation of a disulfide bond between SH1 and SH2, were much faster in scallop S1.ADP than in rabbit skeletal S1.ADP and were rate-limited by the initial attachment of the reagent to scallop S1. The cross-linking sites were mapped by mass spectrometry to SH1 and SH2. These results reveal isoform-specific differences in the conformation and dynamics of the SH1-SH2 helix, providing a possible explanation for destabilization of this helix in some scallop S1 but not in other S1 isoform structures.     

Position of Mercenaria regulatory light-chain Cys50 site on the surface of myosin visualized by electron microscopy

Mercenaria regulatory light-chains, specifically labelled at cysteine 50 with N-iodoacetyl-N'-biotinylhexylenediamine, were rebound to regulatory light-chain denuded scallop myosin, and the hybrid myosin formed was decorated with avidin. These hybrid myosins were visualized by rotary-shadowing electron microscopy. Three distinct images of avidin-decorated hybrid myosin molecules were obtained. These comprise singly decorated molecules, where the avidin is bound symmetrically or asymmetrically with respect to the two heads of myosin, in addition to "figures-of-five", where two myosin molecules associate with a centrally placed avidin molecule. Analysis of these images indicates that the Mercenaria regulatory light-chain Cys50 site is located 15 to 35 A from the head-rod junction when the light-chain is bound in situ to myosin. Implications with respect to head topology and probe studies are discussed.