Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, K_v4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca^2 +DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS–DREAM complexes increases upon Ca^2 + association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca^2 +DREAM were determined to be 195 ± 20 μM and 62 ± 4 μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of K_v4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.     

Molecular mechanism of the P-type ATPases

The recent determination of the structure of the Ca²⁺-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca²⁺-bound state and to near atomic resolution in the Ca²⁺-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 Å away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1

2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg²⁺ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi > ADP·AlF₄⁻ > ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.     

Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle

The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeF_x complex (stable analog of S1¹-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeF_x complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.     

Structural rearrangements in the active site of smooth-muscle myosin

Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 A and 24.9 A, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-A closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.     

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.     

Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system

One of the reactive thiols in the myosin head, SH1, was covalently labeled with a biotin derivative, N-iodoacetyl-N'-biotinylhexylenediamine. When 50% of the SH1 thiol was modified with the biotin reagent as judged from measurements of ATPase activities, the biotinylated myosin bound one mole of avidin per mole of myosin at the saturating level. The avidin-myosin complex was readily formed in the presence of MgADP or MgATP. Peptide maps of the biotinylated myosin revealed that SH1 is actually the site of biotinylation with N-iodoacetyl-N'-biotinylhexylenediamine. Electron microscopic examination of the avidin-myosin complex showed that the attachment site of avidin on the myosin head is 130 A from the head-rod junction, indicating that the SH1 thiol is located there.     

Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

Use of specific lysine modifications to locate the reaction site of cytochrome c with cytochrome oxidase.

The reaction of cytochrome c with trifluoromethylphenyl isocyanate was carried out under conditions which led to the modification of a small number of the 19 lysines. Extensive ion-exchange chromatography was used to separate and purify six different derivatives, each modified at a single lysine residue, lysines 8, 13, 27, 72, 79, and 100, respectively. The only modifications which affected the activity of cytochrome c with cytochrome oxidase (EC 1.9.3.1) were those of lysines immediately surrounding the heme crevice, lysines 13, 27, 72, and 79, and also lysine 8 at the top of the heme crevice. In each case, the modified cytochrome c had the same maximum velocity as that of native cytochrome c, but an increased Michaelis constant for high affinity phase of the reaction. This supports the hypothesis that the cytochrome oxidase reaction site is located in the heme crevice region, and the highly conserved lysine residues surrounding the heme crevice are important in the binding.     

Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.     

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca²⁺-binding properties of the light chains. The Ca²⁺-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH₂ thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca²⁺-binding properties of light chains and generation of tension. When the SH₂ moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca²⁺-binding properties of light chain LC₂ is lost; under these conditions the Ca²⁺-binding affinity value of SH₂-N-ethylmaleimide-blocked myosin (3.3×10⁴m⁻¹) decreases to near that expressed with the dissociated light chain LC₂ (0.7×10⁴m⁻¹). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH₂ thiol does not affect Ca²⁺ binding. The native secondary and tertiary structure of myosin seem to be required for Ca²⁺ binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH₂-N-ethylmaleimide-blocked myosin normal Ca²⁺- and (Mg²⁺+actin)-stimulated ATPase activities are expressed; however, there is a loss in K⁺-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca²⁺ with alterations in pH values. In the absence of Ca²⁺/EGTA buffer the biphasic Ca²⁺-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2×10⁶m⁻¹ and site two: 0.4×10⁶m⁻¹) as compared with values obtained at pH6.5 (site one: 0.64×10⁶m⁻¹ and site two: 0.2×10⁶m⁻¹). The Ca²⁺-binding affinity of light chain LC₂ and S₁, where the (S-1)–(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca²⁺-binding affinity, approx. 0.7×10⁴m⁻¹, whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca²⁺-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S₁ myosin subfragment light chain LC₂ was lost and thus was added back to the purified S₁ fraction. Light chain LC₂ was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)–(S-2) junction is needed for the positioning of light chain LC₂ and thus influences its essential conformation for Ca²⁺ binding.     

Deletion of the myopathy loop of Dictyostelium myosin II and its impact on motor functions

One of the putative actin-binding sites of Dictyostelium myosin II is the beta-strand-turn-beta-strand structure (Ile(398)-Leu-Ala-Gly-Arg-Asp(403)-Leu-Val(405)), the "myopathy loop, " which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg(397)), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp(403)), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile(398), Leu(399), Leu(404), and Val(405)), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp(403) or Arg(402) with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Ca-ATPase structure in the E1 and E2 conformations: mechanism, helix-helix and helix-lipid interactions

The determination of the crystal structure of the Ca²⁺-ATPase of sarcoplasmic reticulum (SR) in its Ca²⁺-bound [Nature 405 (2000) 647] and Ca²⁺-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca²⁺-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM α-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 Å, rather than the normal 30 Å. The experimentally determined TM α-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM α-helices in the Ca²⁺-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM α-helices, with changes in TM α-helical packing being important in the process of transfer of Ca²⁺ ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM α-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca²⁺-ATPase.     

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.     

The Ca/Mg sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils

The Ca²⁺/Mg²⁺ sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca²⁺ was bound to the N-terminal regulatory Ca²⁺-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca²⁺. NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1 mM or 30 μM free Mg²⁺. ATPase activity was maximal when sites III and IV were occupied by Mg²⁺ and it steadily declined as Ca²⁺ displaced Mg²⁺. The data suggest that in the absence of Ca²⁺ at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg²⁺ rather than Ca²⁺. This finding could be relevant to the contraction–relaxation kinetics of cardiac muscle. As Ca²⁺ dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca²⁺ bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.     

Dissociation and reassociation of rabbit skeletal muscle myosin

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25°) and in the obsence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4°C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5–10 per cent release of alkali light chains, LC₁ and LC₃, and a 50 per cent dissociation of the Ca²⁺ binding light chain, LC₂, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15–20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca²⁺ binding sites.