Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head.

The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.

A novel small protein associated with a conjugated trienoic chromophore from membranes of scallop adductor muscle: phosphorylation by protein kinase A.

Membranes enriched in sarcolemma from the cross-striated adductor muscle of the deep sea scallop have been found to contain a previously undescribed small protein of 6-8 kDa that can be released by treatment with organic solvent mixtures. This proteolipid co-purified with a non-amino acid chromophore containing a conjugated trienoic moiety. Although common in plants and algae, such a stable conjugated trienoic group is unusual for an animal cell. The N-terminal amino acid sequence of the protein was XEFQHGLFGXF/ADNIGLQ, which most strongly resembles sequences in the triacyl glycerol lipase precursor and the product of the human breast cancer susceptibility gene BRCA 1, but does not show similarity to previously described proteolipids. The protein was found to be one of the major substrates in its parent membrane for the catalytic subunit of protein kinase A, which may imply a regulatory function for this molecule.

Regulation by molluscan myosins.

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.

Regulation of scallop myosin by calcium. Cooperativity and the "off" state.

Scallop subfragment 1 (S1) is an unregulated molecule; it differs from heavy meromyosin (HMM) and myosin in that it has no "off" state, although it contains the full complement of light chains and the triggering calcium binding site. S1 differs from myosin by lacking the head-rod junction and being single-headed. The contribution of the head-rod junction was evaluated by studying single-headed myosin. Isolated single-headed myosins show some regulation; their actin activated ATPase is stimulated about 3-fold by calcium. However, in contrast to HMM and myosin, the calcium dependence of ATPase activation of single-headed myosin is non-cooperative. The single ATP turnover rate of single-headed myosin in the absence of calcium is less than 30 seconds (our experimental resolution) compared to the approximately 5 minute turnover rate of myosin. HMM and myosin exhibit several cooperative features not shown by S1. Calcium binding becomes cooperative in the presence of nucleotide analogues in HMM and myosin, but not in S1. Nucleotide analogues are bound cooperatively by myosin and HMM in the absence of calcium; the introduction of calcium to the system reduces the affinity and abolishes the cooperative binding of nucleotide in the double headed molecules. Conversely, S1 shows normal binding curves for nucleotide analogues both in the presence and absence of calcium. Therefore, there is direct communication between the calcium binding sites and nucleotide binding sites in regulated molecules that is mediated by interaction between the two heads. .

Cooperativity and regulation of scallop myosin and myosin fragments.

Scallop heavy meromyosin (HMM) preparation obtained by a new improved method showed a Mg-ATPase activity that was activated 15-fold by calcium. The ATPase activity depended on ionic strength and reached maximum at 0.1 M without altering calcium sensitivity. The highly regulated HMM and myosin preparations showed cooperative properties not seen with unregulated subfragment 1 (S1). ATPase activity of myosin and HMM increased steeply with calcium concentration, yielding Hill coefficients about 3 and 4, respectively. Calcium binding by HMM and myosin became cooperative in the presence of ADP, AMP-PNP, or ADP.Vi yielding Hill coefficients of 1.8 and 2.8, respectively. Binding of calcium by HMM in the presence of ATP was also cooperative at physiological ionic strength, whereas at low ionic strength the data fit best to a simple binding curve. In contrast, calcium binding by unregulated S1 followed a normal binding curve and was not affected by the presence of nucleotide analogues. Calcium decreased the affinity of ADP and ADP-PNP to myosin and HMM, but had no effect on the nucleotide binding to S1. The results indicate that communication between the nucleotide and calcium binding sites requires the presence of two heads and exists only in the "off" state. We propose that in the presence of calcium, interaction between the two heads is disrupted and they act independently.

Single-headed scallop myosin and regulation.

Single-headed scallop myosin (shM) was prepared by papain digestion of filamentous scallop myosin and purified by hydrophobic interaction chromatography. The shM preparation consisted of equimolar amounts of polypeptides corresponding to an intact heavy chain, rod chain, essential light chain, and regulatory light chain. In electron micrographs the shape of shM showed the presence of a single head domain to which a normal looking rod was attached. Myosin and shM bound Ca2+ with association constants of 5 x 10(6) and 11 x 10(6) M-1, respectively. The ATPase activity of shM was activated about 3-fold by Ca2+. Both heads of myosin and shM had comparable ATPase activities in the presence of Ca2+. The activation of the ATPase activity of single-headed scallop myosin by Ca2+ paralleled closely the Ca2+ binding, in sharp contrast to the activation of intact myosin by Ca2+, which is highly cooperative. Single turnover experiments of myosin with radioactive ATP gave a half-life for the ATPase cycle of approximately 3 min in the presence of EGTA, whereas that of single-headed myosin was shorter than approximately 30 s, which was the resolution time of these measurements. The results suggest that the presence of two heads, as well as the attachment of the head to the coiled coil rod, contribute to the regulation of scallop myosin by Ca2+.

Sequence variations in the surface loop near the nucleotide binding site modulate the ATP turnover rates of molluscan myosins.

The muscle and species-specific differences in enzymatic activity between Placopecten and Argopecten striated and catch muscle myosins are attributable to the myosin heavy chain. To identify sequences that may modulate these differences, we cloned and sequenced the cDNA encoding the myosin heavy chains of Placopecten striated and catch muscle. Deduced protein sequences indicate two similar isoforms in catch and striated myosins (97% identical); variations arise by differential RNA splicing of five alternative exons from a single myosin heavy chain gene. The first encodes the phosphate-binding loop; the second, part of the ATP binding site; the third, part of the actin binding site; the fourth, the hinge in the rod; and the fifth, a tailpiece found only in the catch muscle myosin heavy chain. Both Placopecten myosin heavy chains are 96% identical to Argopecten myosin heavy chaina isoforms. Because subfragment-1 ATPase activities reflect the differences observed in the parent myosins, the motor domain is responsible for the variations in ATPase activities. In addition, data show that differences are due to Vmax and not actin affinity. The sequences of all four myosin heavy chain motor domains diverge only in the flexible surface loop near the nucleotide binding pocket. Thus, the different ATPase activities of four molluscan muscle myosins are likely due to myosin heavy chain sequence variations within the flexible surface loop that forms part of the ATP binding pocket of the motor domain.

Regulatory domains of myosins: influence of heavy chain on Ca(2+)-binding.

Light chain binding domains of rabbit skeletal, turkey gizzard and scallop myosin comprised of equimolar amounts of a short heavy chain fragment, essential light chain, and regulatory light chain have been obtained following extensive tryptic digestion. These complexes that are analogous to the regulatory domain prepared previously from scallop myosin by digestion with clostripain resist proteolysis due to the mutual protection of the heavy chain and the light chains, and are common structural features of the myosins studied. Specific Ca(2+)-binding by the regulatory domains reflects the behaviour of intact myosin; only scallop regulatory domain has a specific Ca(2+)-binding site. The heavy chain fragments of the different regulatory domains have been isolated under denaturing conditions and reconstituted with scallop essential light chain and scallop regulatory light chain or turkey gizzard regulatory light chain to yield regulatory domain hybrids. Hybrids containing the turkey gizzard regulatory light chain were used in Ca(2+)-binding studies since they were far more stable than their counterparts with the scallop regulatory light chain. The gizzard hybrid binds Ca2+ with a comparable specificity but somewhat lower affinity than native scallop regulatory domain. The rabbit regulatory domain hybrid also binds Ca2+, although with a reduced affinity and specificity. The results indicate that Ca(2+)-binding ability is determined by the light chains and modified by the heavy chains.

Structure of the regulatory domain of scallop myosin at 2.8 A resolution.

The regulatory domain of scallop myosin is a three-chain protein complex that switches on this motor in response to Ca2+ binding. This domain has been crystallized and the structure solved to 2.8 A resolution. Side-chain interactions link the two light chains in tandem to adjacent segments of the heavy chain bearing the IQ-sequence motif. The Ca(2+)-binding site is a novel EF-hand motif on the essential light chain and is stabilized by linkages involving the heavy chain and both light chains, accounting for the requirement of all three chains for Ca2+ binding and regulation in the intact myosin molecule.

Effect of the biochemical state of the Ca-ATPase protein of scallop sarcoplasmic reticulum on its interaction with trans-parinaric acid.

The polyene fluorescent probe trans-parinaric acid (tPA) was used to compare lipid-protein interactions in the scallop fragmented sarcoplasmic reticulum (FSR) between biochemical states where the Ca-ATPase molecules were arranged differently in the membrane and had different tertiary conformations. The state of the bulk lipid phase was examined over the temperature range -3 to +32 degrees C by exciting the tPA directly at 320 nm. The state of the system close to the Ca-ATPase protein was followed over the same temperature range by indirectly exciting the tPA through resonance energy transfer from the Ca-ATPase protein, with approximately one twenty-fifth the quantum yield of the directly excited probe. Raising the tPA/lipid ratio in the membrane to high levels (approx. 1:9), caused the quantum yield of indirectly excited tPA to reach a maximum, which may reflect saturation of the annular lipid phase with the probe, or contribution to the fluorescence from indirectly excited tPA bound directly to the protein. In the presence of 0.1 M KCl, a thermal perturbation was observed at approx. 7 degrees C using indirect excitation when the Ca(2+)-binding sites on the Ca-ATPase were occupied, and the subunits were disorganized. This transition was not detected in the presence of 0.1 M KCl and EGTA, when the Ca(2+)-binding sites were empty, and the Ca-ATPase subunits were organized in dimeric arrays. The transition seen with the E1(Ca2+)2 form of the membrane may involve an event at the protein/lipid interface, or a change in the environment of tPA bound to the Ca-ATPase. The temperature at which the perturbation occurs is close to that of a discontinuity in the Arrhenius plot of the Ca-ATPase enzyme activity determined in the presence of 0.1 M KCl (Kalabokis, V.N. and Hardwicke, P.M.D. (1988) J. Biol. Chem. 263, 15184-15188). No perturbation was observed in the bulk properties of the lipid component of the membrane in either the E1(Ca2+)2 or E2 states.

Effect of Na+ and nucleotide on the stability of solubilized Ca(2+)-free Ca-ATPase from scallop sarcoplasmic reticulum.

In membranous scallop sarcoplasmic reticulum, the alkali metal cations Na+ and K+ and nucleotide together promote dimer formation by the Ca(2+)-free Ca-ATPase and stabilize the enzyme activity [Kalabokis, V. N., Bozzola, J. J., Castellani, L., & Hardwicke, P. M. D. (1991) J. Biol. Chem. 266, 22044-22050]. The dependence of stabilization of the Ca(2+)-free membranous scallop Ca-ATPase on Na+ concentration does not show saturation and may involve several superimposed effects. In order to assess the contribution of dimer toward stabilization, i.e., determine the relative importance of intra- and intermolecular effects on stabilization, the influence of varying Na+ concentration and nucleotide on the decay of enzyme activity of the Ca(2+)-free detergent-solubilized Ca-ATPase was studied. Loss of enzyme activity on removal of Ca2+ with EGTA was associated with loss of capacity for phosphorylation by ATP, a Ca(2+)-dependent function. Stabilization of the soluble Ca(2+)-free enzyme by Na+ showed major differences from that seen with the membranous enzyme. The extent of stabilization of the Ca(2+)-free soluble enzyme by Na+ showed clear saturation with increasing Na+ concentration. In contrast to the Ca(2+)-free membranous enzyme, which is inactivated at pH 7.0 with biphasic first-order kinetics, loss of enzymatic function by the solubilized Ca-ATPase at pH 6.92, 0 degrees C, followed monophasic first-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible role for the dimer ribbon state of scallop sarcoplasmic reticulum. Dimmer ribbons are associated with stabilization of the Ca(2+)-free Ca-ATPase.

The Ca-ATPase activity of membranous scallop sarcoplasmic reticulum was found to be unstable when the Ca(2+)-binding sites on the Ca-ATPase were unoccupied. The decay in activity could be slowed or halted by inclusion in the preincubation medium of Na+, K+, nucleotides, ethylene glycol, or high concentrations of choline chloride. Stabilization of the Ca(2+)-free Ca-ATPase by Na+ and K+ showed a markedly different concentration dependence to that seen with activation of the Ca(2+)-activated ATPase activity by the two ions. Examination in the electron microscope of scallop membranes negatively stained in the presence of EGTA under conditions where the enzyme had been stabilized against lack of Ca2+ always showed vesicles containing dimer ribbon structures, whereas unstabilized membranes did not show dimer ribbons. There was an association between the effectiveness of a medium in stabilizing the enzyme in the presence of EGTA and the extent and quality of the dimer arrays seen in the microscope. Comparison of the range of Ca2+ concentration over which the Ca(2+)-binding sites on the scallop Ca-ATPase titrated with the range over which the dimer ribbon structural state was lost indicated that the Ca(2+)-binding sites on the Ca-ATPase must be empty for dimer ribbon formation to occur. Previous studies (Franzini-Armstrong, C., Ferguson, D. G., Castellani, L., and Kenney, L. J. (1987) Ann. N. Y. Acad. Sci. 483, 44-56) have found that the Ca-ATPase molecules in scallop adductor muscle freeze-fractured after fixation under relaxing conditions are arranged in dimer ribbons. Thus, the association of stabilization of the Ca(2+)-free Ca-ATPase with the presence of dimer ribbons implies that one function of the dimer state may be to stabilize the scallop enzyme in situ, when the Ca2+ concentration in the sarcoplasm is low and the muscle is relaxed.