Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides.

We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor domain (S-1) as actin site-directed agents and label exclusively and stoichiometrically Cys 540 in the hydrophobic strong actin binding helix-loop-helix motif, causing only a 1.9-2.4-fold decrease in the V(max) for acto-S-1 ATPase. The covalently attached cysteaminyl probe side chain spans maximally 17 and 8 A, respectively, and the fluorophores have different polarity, volume, and flexibility. Thus, they may provide complementary spectroscopic information on the environmental properties of this critical actin binding region. Here, we have analyzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluorescein label or with the Tb(3+) or Eu(3+) chelate of the other label to assess the conformational transitions precisely occurring at this site upon interaction with F-actin, nucleotides, or phosphate analogues. For either label, specific spectral changes of significant amplitude were obtained, identifying at least two major structural states. One was mediated by rigor binding of F-actin in the absence or presence of MgADP. It was abolished by MgATP, and it was not produced by the binding of nonpolymerizable G-actin. A modeling of the corresponding changes in the intensity and lambda(max) of the fluorescence emission spectra, achieved using the fluorescent adducts of 2-mercaptoethanol in varying concentrations of dimethylformamide, illustrates the predicted apolar nature of the strong acto-S-1 interface. A second state was promoted by the binding of ATP, AMP-PNP, ADP.AlF4, ADP. BeFx, or PP(i). It should be prevalent in the weak acto-S-1 binding complexes. The accompanying fluorescence intensity reduction, observed with each label, in both the absence and presence of F-actin, would result from a specific modification by these ligands of the probe orientation and/or solvent accessibility as suggested by acrylamide quenching experiments. It could represent the spectral manifestation of the predicted allosteric linkage from the ATPase site to the strong actin binding site of S-1 that modulates the acto-S-1 affinity. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospecific and hydrophobic actin binding motif during the skeletal cross-bridge cycle.

An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms.

Smooth muscle myosin isoforms of the heavy chain and the essential light chain have been hypothesized to contribute to the different shortening velocities of phasic and tonic smooth muscles, and to their different affinities for MgADP. We used the baculovirus/insect cell system to express homogeneous heavy meromyosin molecules differing only in seven amino acid insert (QGPSFSY) in the motor domain near the active site, or in the type of essential light chain isoform. Myosin from tonic rabbit uterine smooth muscle lacks the heavy chain insert, while myosin from phasic chicken gizzard contains it. The properties of a mutant uterine heavy meromyosin with added insert, and a mutant gizzard heavy meromyosin with the insert deleted, were compared with their wild type progenitors. Phosphorylated heavy meromyosins with the insert have a twofold higher enzymatic activity and in vitro motility han heavy meromyosins without the insert. These functional properties were not altered by the essential light chain isoforms. The altered motility caused by the insert implies that it modulates the rate of ADP release, the molecular step believed to limit shortening velocity. The insert may thus account in part for both the lower sensitivity to MgADP and the higher shortening velocity of phasic compared to tonic smooth muscles.

Deletion of amino acids from the carboxy-terminal end of actin.

A series of deletions was made from the C-terminal end of actin by inserting termination codons into a full length cDNA of human alpha-skeletal muscle actin. These included deletions of 2, 3, 10, 20, 30, and 40 amino acids. The cDNA clones were transcribed and the resulting mRNAs were translated in vitro using 35S-labeled methionine. The 35S-labeled actin and actin mutants were then tested for the ability to coassemble with carrier actin, bind DNAse I, bind myosin S-1, bind a 27 kDa proteolytic fragment of alpha-actinin, and incorporate into myofibrils in vitro. Removal of the C-terminal two or three amino acids did not grossly alter the properties of actin tested. Deletion of an additional 7 amino acids (10 amino acids total) significantly decreased coassembly, binding to DNAse I, and incorporation into myofibrils, but did not dramatically reduce binding to myosin S-1 or the 27 kDa fragment of alpha-actinin. Deletion of 20 or more amino acids virtually abolished all normal actin function tested. By examining the structure of actin, we propose that the effect of removing residues 356-365 is due to the important role Trp356 plays in maintaining hydrophobic bonds between three non-contiguous segments of actin. We also suggest that removal of residues 366-372 adversely affected the structure or orientation of the DNAse I binding loop and that this change can account for defects in actin binding to DNAse I, coassembly with wild type actin, and incorporation into myofibrils.

Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli and their purification and characterisation. Two point mutations allow high-level expression of troponin-I.

We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG-->GCC and (Gly4) GGG-->GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin-C. Both proteins were purified using ion-exchange chromatography and have been proved to be biologically active. The recombinant troponin-I was able to bind to a troponin-C affinity column in the presence of 9 M urea in a calcium-dependent manner. The calcium-dependent troponin-I-troponin-C complex between both recombinant proteins was also demonstrated by alkaline-urea gel electrophoresis. In addition, troponin-I inhibited the acto-S1 Mg-ATPase activity; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin-C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium-dependent force generation to calcium-insensitive skinned muscle fibres.

Structural study of gizzard caldesmon and its interaction with actin. Binding involves residues of actin also recognised by myosin subfragment 1.

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1-44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389-397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20-41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483-578) derived from the N-terminal portion of a 35-kDa proteolytic cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin-filament regulation in smooth and striated muscle.

Normal expression of myosin fast alkali light chain 3 in the hindlimb muscle of chick embryos paralyzed with curare.

We have used a monoclonal antibody (Mab) raised against the fast alkali light chains of quail pectoral muscle myosin to study the expression of MLC1f and MLC3f in the hindlimb muscle of a staged series of control chick embryos and 16-day embryos that had been paralyzed with curare. The Mab (QBM-2) is highly specific for the fast myosin alkali light chains of chick, hamster, and human muscle myosin. On Western blots, MLC1f is detected in hindlimb actomyosin at all of the stages examined, whereas MLC3f cannot be detected until Embryonic Day 14 (E14). Most of the E16 embryos that had been paralyzed with curare since E4 express detectable levels of both MLC1f and MLC3f in their hindlimb muscles, even though embryos incubated under these conditions do not exhibit spontaneous limb movements. Contrary to other reports, our results demonstrate that muscle contraction is not required for the accumulation of MLC3f. In light of our previous finding that innervation is essential for MLC3f accumulation in limb buds grafted onto the chorioallantoic membrane of chick hosts, these results suggest that some neural influence other than contraction, possibly a trophic factor, may play a role in the developmentally regulated expression of MLC3f in avian limb muscle.

Inhibition of actomyosin subfragment 1 ATPase activity by analog peptides of the actin-binding site around the Cys(SH1) of myosin heavy chain.

The synthetic heptapeptide, Ile-Arg-Ile-Cys-Arg-Lsy-Gly-ethoxy, an analog of one of the actin binding sites on myosin head (S-site) (Suzuki, R., Nishi, N., Tokura, S., and Morita, F. (1987) J. Biol. Chem. 262, 11410-11412) was found to completely inhibit the acto-S-1 (myosin subfragment 1) ATPase activity. The effect of the heptapeptide on the binding ability of S-1 for F-actin was determined by an ultracentrifugal separation. Results indicated that the heptapeptide scarcely dissociated the acto-S-1 complex during the ATPase reaction. Consistent results were obtained from the acto-S-1 ATPase activities determined as a function of S-1 concentrations in the absence or presence of the heptapeptide at a fixed F-actin concentration. The heptapeptide reduced the maximum acto-S-1 ATPase activity without affecting the apparent dissociation constant of the acto-S-1 complex. The heptapeptide bound by a site on actin complementary to the S-site probably inhibits the activation of S-1 ATPase by F-actin. These results suggest that S-1 ATPase is necessary to rebind transiently with F-actin at the S-site in order to be activated by F-actin. This is consistent with the activation mechanism proposed assuming the two actin-binding sites on S-1 ATPase (Katoh, T., and Morita F. (1984) J. Biochem. (Tokyo) 96, 1223-1230).

Interaction between stretch of residues 633-642 (actin binding site) and nucleotide binding site on skeletal myosin subfragment 1 heavy chain.

Using a complementary sequence or antipeptide to selectively neutralize the stretch of residues 633-642 of skeletal myosin heavy chain, we recently demonstrated that this segment is an actin binding site operating in the absence as in the presence of nucleotide and that this stretch 633-642 is not part of the nucleotide binding site [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475]. In the present study, we determined that the covalent cross-linking of the antipeptide to the stretch 633-642 [induced by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] does not alter the overall polypeptide conformation since no changes were observed on the far-ultraviolet CD spectra and thiol reactivity measurements. The presence of the antipeptide did not influence significantly the enhancement of tryptophan fluorescence induced by ATP.Mg2+ or ADP.Mg2+ binding to the myosin head (S1) nor did it on the ATP.Mg2+-induced tryptic proteolysis of S1 heavy chain. Moreover, fluorescence quenching studies, using acrylamide and the analogue, 1,N6-ethenoadenosine 5'-triphosphate, indicated that the nucleotide bound to antipeptide-S1 complex has an accessibility to the solute quencher close to that observed when it is bound to native S1. Additionally, neutralization of the stretch 633-642 of the S1 heavy chain by the antipeptide did not influence the stabilization of the Mg2+.ADP.sodium vanadate-S1 complex. On the other hand, experiments using antipeptide-induced protection against the cleavage of the S1 heavy chain by Arg-C protease demonstrated that the presence of Mg2+.ADP.sodium vanadate in the S1 nucleotide site did not affect the interaction of the antipeptide with the stretch of residues 633-642.(ABSTRACT TRUNCATED AT 250 WORDS)

Desensitization of substrate inhibition of acto-H-meromyosin ATPase by treatment of H-meromyosin with rho-chloromercuribenzoate. Relation between the extent of desensitization and the amount of bound rho-chloromercuribenzoate1.

H-Meromyosin (CMB leads to betaME-H-meromyosin) was prepared by tryptic digestion of myosin, which had been treated with CMB bound to H-meromyosin and the extent of desensitization of the substrate inhibition of acto-H-meromyosin ATPase [EC 3.6.1.3.] was investigated. Both the dissociation of acto-H-meromyosin induced by ATP and substrate inhibition decreased with increase in the amount of bound CMB to a minimum value at about 1 mole of CMB bound per mole of H-meromyosin. The substrate inhibition of acto-H-meromyosin ATPase was restored to the original level by complete removal of the bound CMB by further treatment of CMB leads to beta ME-H-meromyosin with a large excess of beta-mercaptoethanol. The dissociation constant of acto-H-meromyosin in the presence of ATP decreased markedly on modification with CMB, while the maximum ATPase activity ar a sufficiently high concentration of F-actin remained essentially unchanged. Acto-H-meromyosin was reconstituted from F-actin and CMB LEADS TO beta ME-H-meromyosin, containing less than the stoichiometric amount of bound CMB. Its ATPase activity and the extent of dissociation of acto-H-meromyosin induced by ATP were explained as those of a mixture of unmodified H-meromyosin and CMB leads to beta ME-H-meromyosin containing 1 mole of CMB per mole of H-meromyosin. Half of the light chains (g2), with a molecular weight of 18,000, were removed from myosin by treatment with CMB and beta-mercaptoethanol. After this treatment, on further incubation of the myosin with a large excess of beta-mercaptoethanol, the myosin contained only half of the g2, but the substrate inhibition of acto-H-meromyosin ATPase was restored completely. The initial burst of P1 liberation and the EDTA-ATPase activity decreased to almost zero on specific modification of the SH1-groups with NEM, while the initial burst decreased to some extent and the EDTA-ATPase activity to 50% of the original value on binding of 1 mole CMB per mole of H-meromyosin. The actomyosin-type of ATPase activity was strongly inhibited by modification with CMB. The extent of the dissociation of acto-H-meromyosin induced by ATP was unaffected by modification with NEM, while it decreased on further treatment of NEM-myosin with CMB FOLLOWED BY BETA-MERCAPTOETHANOL.