Purification, crystallization and preliminary X-ray crystallographic analysis of squid heavy meromyosin.

All muscle-based movement is dependent upon carefully choreographed interactions between the two major muscle components, myosin and actin. Regulation of vertebrate smooth and molluscan muscle contraction is myosin based (both are in the myosin II class), and requires the double-headed form of myosin. Removal of Ca2+ from these muscles promotes a relatively compact conformation of the myosin dimer, which inhibits its interaction with actin. Although atomic structures of single myosin heads are available, the structure of any double-headed portion of myosin, including the ∼375 kDa heavy meromyosin (HMM), has only been visualized at low (∼20 Å) resolution by electron microscopy. Here, the growth of three-dimensional crystals of HMM with near-atomic resolution (up to ∼5 Å) and their X-ray diffraction are reported for the first time. These crystals were grown in off-state conditions, that is in the absence of Ca2+ and the presence of nucleotide analogs, using HMM from the funnel retractor muscle of squid. In addition to the crystallization conditions, the techniques used to isolate and purify this HMM are also described. Efforts at phasing and improving the resolution of the data in order to determine the structure are ongoing.

Crystal structure of a phosphorylated light chain domain of scallop smooth-muscle myosin.

We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg(16), an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

Myosin cleft closure determines the energetics of the actomyosin interaction.

Formation of the strong binding interaction between actin and myosin is essential for force generation in muscle and in cytoskeletal motor systems. To clarify the role of the closure of myosin's actin-binding cleft in the actomyosin interaction, we performed rapid kinetic, spectroscopic, and calorimetric experiments and atomic-level energetic calculations on a variety of myosin isoforms for which atomic structures are available. Surprisingly, we found that the endothermic actin-binding profile of vertebrate skeletal muscle myosin subfragment-1 is unique among studied myosins. We show that the diverse propensity of myosins for cleft closure determines different energetic profiles as well as structural and kinetic pathways of actin binding. Depending on the type of myosin, strong actin binding may occur via induced-fit or conformational preselection mechanisms. However, cleft closure does not directly determine the kinetics and affinity of actin binding. We also show that cleft closure is enthalpically unfavorable, reflecting the development of an internal strain within myosin in order to adopt precise steric complementarity to the actin filament. We propose that cleft closure leads to an increase in the torsional strain of myosin's central ß-sheet that has been proposed to serve as an allosteric energy-transducing spring during force generation.

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin.

We have determined the 2.3-Å-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ("smooth") muscle. This structure reveals hinges that may function in the "on" and "off" states of myosin. The molecule adopts two different conformations about the heavy chain "hook" and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 Å. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

The ultrastructure and contractile properties of a fast-acting, obliquely striated, myosin-regulated muscle: the funnel retractor of squids.

We investigated the ultrastructure, contractile properties, and in vivo length changes of the fast-acting funnel retractor muscle of the long-finned squid Doryteuthis pealeii. This muscle is composed of obliquely striated, spindle-shaped fibers ~3 mum across that have an abundant sarcoplasmic reticulum, consisting primarily of membranous sacs that form 'dyads' along the surface of each cell. The contractile apparatus consists of 'myofibrils' approximately 0.25-0.5 microm wide in cross section arrayed around the periphery of each cell, surrounding a central core that contains the nucleus and large mitochondria. Thick myofilaments are approximately 25 nm in diameter and approximately 2.8 microm long. 'Dense bodies' are narrow, resembling Z lines, but are discontinuous and are not associated with the cytoskeletal fibrillar elements that are so prominent in slower obliquely striated muscles. The cells approximate each other closely with minimal intervening intercellular connective tissue. Our physiological experiments, conducted at 17 degrees C, showed that the longitudinal muscle fibers of the funnel retractor were activated rapidly (8 ms latent period following stimulation) and generated force rapidly (peak twitch force occurred within 50 ms). The longitudinal fibers had low V(max) (2.15 +/-0.26 L(0) s(-1), where L(0) was the length that generated peak isometric force) but generated relatively high isometric stress (270+/-20 mN mm(-2) physiological cross section). The fibers exhibited a moderate maximum power output (49.9 W kg(-1)), compared with vertebrate and arthropod cross striated fibers, at a V/V(max) of 0.33+/-0.044. During ventilation of the mantle cavity and locomotion, the funnel retractor muscle operated in vivo over a limited range of strains (+0.075 to -0.15 relative to resting length, L(R)) and at low strain rates (from 0.16 to 0.91 L(R) s(-1) ), corresponding to a range of V/V(max) from 0.073 to 0.42. During the exhalant phase of the jet the range of strains was even narrower: maximum range less than +/-0.04, with the muscle operating nearly isometrically during ventilation and slow, arms-first swimming. The limited length operating range of the funnel retractor muscles, especially during ventilation and slow jetting, suggests that they may act as muscular struts.

The on-off switch in regulated myosins: different triggers but related mechanisms.

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long alpha-helical "heavy chain" stabilized by a "regulatory" light chain (RLC) and an "essential" light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca(2+) to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca(2+). Our results indicate that loss of Ca(2+) abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding.

Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-A x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm =45 degrees to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called "front door." In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-A nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.

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.

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.

Interactions of the two heads of scallop (Argopecten irradians) heavy meromyosin with actin: influence of calcium and nucleotides.

We recently proposed a co-operative model for the influence of calcium and ADP on scallop ( Argopecten irradians ) muscle heavy meromyosin (scHMM), in which scHMM exists in two conformations (designated 'off' and 'on'), and calcium and ADP are allosteric effectors of the equilibrium between the off and on conformations [Nyitrai, Szent-Gyorgyi and Geeves (2002) Biochem. J. 365, 19-30]. Here we examine the influence of actin on scHMM. In the absence of nucleotide, both heads of scHMM bind very tightly to actin, independent of the presence of calcium. In the absence of calcium, ADP dissociates scHMM from actin completely, and little evidence of ternary complex formation can be found (actin affinity >20 microM). The off state of scHMM therefore does not interact with actin. In the presence of calcium, ADP and actin lower each other's affinity for scHMM by 30-50-fold, although both heads remain strongly attached to actin (actin affinity 0.17 microM). Detailed analysis suggests that the second head contributes far more to the overall binding energy than is the case for mammalian skeletal muscle HMM. This is consistent with a different stereochemical relationship between the two heads in scallop and mammalian HMM molecules.

A kinetic model of the co-operative binding of calcium and ADP to scallop (Argopecten irradians) heavy meromyosin.

Analysis of the kinetics of ATP and ADP binding to scallop (Argopecten irradians) heavy meromyosin (HMM) showed that the only calcium-dependent process is the rate of ADP release. At physiological ionic strength calcium accelerated ADP release about 20-fold. Notably in the absence of calcium only one ADP bound HMM, with an affinity of 0.5-1 microM. The second nucleotide site remained unoccupied at up to 50 microM ADP yet could bind ATP rapidly. The calcium dependence of ADP-release rates showed that calcium binds co-operatively to scallop HMM with an affinity of 0.78 microM and a Hill coefficient of 1.9. Detailed interpretation of the data suggests that HMM exists in equilibrium between the on and off states and that calcium and ADP modulate the equilibrium between the two states. The on state is favoured in the presence of calcium and in the absence of both calcium and nucleotide. The off state is favoured by ADP (or ADP * P(i)) in the absence of calcium. A detailed co-operative model of the interaction of ADP and calcium with HMM is presented.