Subfragment 1 Research Articles

Incorporation of an Azobenzene Derivative into the Energy Transducing Site of Skeletal Muscle Myosin Results in Photo-Induced Conformational Changes

The region containing reactive cysteines, Cys 707 (SH1)-Cys 697 (SH2), of skeletal muscle myosin is thought to play a key role in the conformational changes of the myosin head during force generation coupled to ATP hydrolysis. In the present study, we synthesized a photochromic crosslinker, 4,4'-azobenzene-dimaleimide (ABDM), that undergoes reversible cis-trans isomerization upon ultra violet (UV) and visible (VIS) light irradiation resulting in a change in the crosslinking length from 5 to 17 A. The reactive cysteines, SH1 and SH2, of myosin subfragment 1 (S1) were crosslinked with ABDM, yielding an ABDM-S1 complex. The changes in absorbance induced by UV/VIS light irradiation of the complex were similar to those of free ABDM indicating that the incorporation of ABDM at the SH1 and SH2 sites did not disrupt the isomerization of crosslinked ABDM. Small-angle synchrotron X-ray scattering analysis of the ABDM-S1 complex in solution suggested that the localized conformational changes resulting from the cis to trans isomerization on ABDM crosslinking of SH1 and SH2 induced a small but significant swing in the lever arm portion of S1 in the opposite direction from that induced by ATP.

High flexibility of the actomyosin crossbridge resides in skeletal muscle myosin subfragment-2 as demonstrated by a new single molecule assay

Popular views of force generation in muscle indicate that a lever arm in the myosin head initiates displacement of the thin filament. However, this lever arm is attached to the thick filament backbone by a flexible combination of coiled coils and hinges in the myosin subfragment-2 (S2); therefore, efficient force generation depends on tension development in this linking structure. Herein, a single molecule assay is developed to examine the flexibility of the intact S2 relative to that of the myosin head. Fluorescently labeled myosin rod is polymerized onto a single myosin molecule that is bound to actin, and the resulting Brownian motion of the rod is analyzed at video rates by digital image processing. Complete rotations of the rod suggest significant amounts of random coil in the linking structure. The close similarity of twist rates for double-headed and single-headed myosin indicates that most of the flexibility originates at or beyond the first pitch of coiled coil in S2 and most likely at the hinge connecting S2 and the light meromyosin. The myosin head has a smaller but still detectable impact on this flexibility, since the addition of ADP to the rigor crossbridge produces differential effects on the torsional characteristics of double-headed versus single-headed myosin.

Denaturation of Tilapia Myosin Fragments by High Hydrostatic Pressure

ABSTRACT: Hydrostatic pressures (50 to 300 MPa) were applied at 0 °C for 50 min to determine the aggregation and viscoelastic properties of tilapia (Orechromis niloticus) myosin fragments: subfragment‐1 (S‐1) and rod. With pressure lower than 150‐MPa treatment, S‐1 underwent intermolecular interaction to increase its elastic properties. After a 200‐MPa treatment, S‐1 unfolded, aggregated, and decreased its solubility. A turbid solution was obtained after pressurization over 200 MPa. Further, S‐1 transformed to a more elastic gel with pressure increasing. At 200 MPa, S‐1 denatured entirely with no change of enthalpy detected by differential scanning calorimetry (DSC). However, all properties of rod did not change during pressurization. This study reveals that S‐1 contributes the initiation of gel formation in myosin, and the role of rod is not clear. Keywords: hydrostatic pressure, S‐1, rod, denaturation

Expression of a nonpolymerizable actin mutant in Sf9 cells.

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.

Intensity of X-ray reflections from skeletal muscle thin filaments partially occupied with myosin heads: effect of cooperative binding

For quantitative analysis of contractile proteins of muscle by means of X-ray diffraction, it is important to know how the intensities of individual reflections are related to the number of diffracting objects, i.e., the amount of constituent contractile protein in the muscle cell. Here we diffused various amounts of exogenous myosin subfragment-1 (S1) into overstretched skinned skeletal muscle fibers, either in the presence or absence of Ca2+ , and derived the relationship between the S1 content and the intensities of reflections arising from the S1. In theory, the intensities should be proportional to the square of the S1 content (square law). However, the intensity-content relation deviated systematically from the square law as the S1 content was lowered, and it was better described as a linear function at the lower end of the S1 contents (<20% of saturation level). Model calculations show that the way of deviation is explained by the cooperative manner of S1 binding to the regulated thin filament.

The Elongation and Contraction of Actin Bundles are Induced by Double-headed Myosins in a Motor Concentration-dependent Manner

Many types of myosin have been found and characterized to date, and already nearly 20 classes have been identified. However, these myosin motors can be classified more simply into two groups according to their head-structure, i.e. double- or single-headed myosins. Why do some myosin motors possess a double-headed structure? One obvious possible reason would be that the two heads improve the motor's processivity and sliding performance. Previously, to investigate the possibility that the double-headed myosins simultaneously interact with parallel arrayed two actin filaments in the presence of Mg-ATP, we developed an in vitro assay system using actin bundles formed by inert polymers. Using that system, we show here that skeletal muscle heavy meromyosin (HMM), a double-headed myosin derivative, but not subfragment-1 (S-1), a single-headed one, was able to contract or elongate actin bundles in a concentration-dependent manner. Similar elongation or contraction of actin bundles can also be induced by other double-headed myosin species isolated in the native state from Dictyostelium, from green algae Chara or from chicken brain. The results of this study confirm that double-headed myosin motors can induce sliding movements among neighboring actin filaments. The double-headed structure of myosins may also be important for generating tension or elongation in actin bundles or gels, and for organizing polarity-sorted actin networks, not just for improving their motor processivity or activity.

Behavior of caldesmon upon interaction of thin filaments with myosin subfragment 1 in ghost fibers

Caldesmon is a component of smooth muscle thin filaments which inhibits their interaction with myosin. We have used polarized fluorescence technique to study the behavior of caldesmon during the interaction of myosin subfragment 1 (S1) with thin filaments reconstituted in rabbit skeletal muscle ghost fibers by incorporation of smooth muscle tropomyosin and caldesmon labeled with acrylodan at cysteine residue located in the C-terminal region. Significant changes in acrylodan fluorescence intensity upon addition of skeletal muscle S1 reflected substantial displacement of caldesmon from thin filaments, while alterations in the calculated fluorescence parameters indicated the simultaneous rearrangement of the remaining caldesmon fraction. The orientation of caldesmon in the S1-thin filament complex relative to the fiber axis changes by approximately 7° and the mobility of the fluorescent probe by about 9%. The alterations in caldesmon orientation were proportional to the strength of S1 binding and diminished respectively upon addition of ADP and ADP-Vi. The changes in orientation of acrylodan-caldesmon evoked by the interaction of S1 with thin filaments were more pronounced than that in AEDANS-F-actin which suggests that the spatial arrangement of caldesmon in the complex is governed not only by F-actin but also by S1. The results may indicate that the changes in spatial arrangement of caldesmon are adjusted to the conformation of F-actin and S1 characteristic for particular steps of the ATP hydrolysis cycle.

Thymol: a classical small-molecule compound that has a dual effect (potentiating and inhibitory) on myosin

The effect of thymol on the ATPase activity of myosin subfragment-1 (S1) and on the contractile properties of skinned skeletal muscle fibers was studied. At concentrations of 1.5–2 mM, thymol activated the S1 ATPase substantially and the actin-activated S1 ATPase modestly. At the same concentrations, the isometric force of skinned skeletal muscle fibers was modestly suppressed (11% at 2 mM). However, the kinetic parameters of contraction were suppressed more: the velocity of shortening and the rate of force redevelopment after shortening were suppressed by 43% and 31% at 2 mM, respectively. Thus, among other small-molecule inhibitors, thymol is unique in that it has opposite effects on the enzymatic activity and kinetic parameters of contraction. Thymol may serve as a potent tool for studying the mechanism of coupling between the ATPase reaction and contraction in muscle.

Thymol: a classical small-molecule compound that has a dual effect (potentiating and inhibitory) on myosin

The effect of thymol on the ATPase activity of myosin subfragment-1 (S1) and on the contractile properties of skinned skeletal muscle fibers was studied. At concentrations of 1.5–2 mM, thymol activated the S1 ATPase substantially and the actin-activated S1 ATPase modestly. At the same concentrations, the isometric force of skinned skeletal muscle fibers was modestly suppressed (11% at 2 mM). However, the kinetic parameters of contraction were suppressed more: the velocity of shortening and the rate of force redevelopment after shortening were suppressed by 43% and 31% at 2 mM, respectively. Thus, among other small-molecule inhibitors, thymol is unique in that it has opposite effects on the enzymatic activity and kinetic parameters of contraction. Thymol may serve as a potent tool for studying the mechanism of coupling between the ATPase reaction and contraction in muscle.

Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

Myosin-Induced Movement of αα, αβ, and ββ Smooth Muscle Tropomyosin on Actin Observed by Multisite FRET

The interaction of the αα, ββ, and αβ smooth muscle tropomyosin (Tm) isoforms with F-actin was systematically studied in the absence and in the presence of myosin subfragment 1 (S1) using multifrequency phase/modulation Förster resonance energy transfer (FRET). A Gaussian double distance distribution model was adopted to fit FRET data between a 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid donor at either Cys-36 of the β-chain or Cys-190 of the α-chain and a 4-dimethylaminophenylazophenyl 4′-maleimide acceptor at Cys-374 of F-actin. Experimental data were obtained for singly and doubly labeled αβ Tm (donor only at α, only at β, or both) and for doubly labeled αα or ββ Tm. Data for singly labeled αβTm were combined in a global analysis with doubly labeled αβTm. In all doubly labeled isoforms, upon S1 binding, one donor-acceptor “apparent” distance increased slightly by 0.5–2 Å, whereas the other decreased by 6–9 Å. These changes are consistent with a uniform “rolling” motion of Tm over the F-actin surface. The analysis indicates that Tm occupies relatively well-defined positions, with some flexibility, in both the predominantly closed (−S1) and open (+S1) thin-filament states. The results for the αβTm heterodimer indicate that the local twofold symmetry of αα or ββ Tm is effectively broken in αβTm bound to F-actin, which implies a difference between the α- and β-chains in terms of their interaction with F-actin.

Behavior of caldesmon upon interaction of thin filaments with myosin subfragment 1 in ghost fibers

Caldesmon is a component of smooth muscle thin filaments which inhibits their interaction with myosin. We have used polarized fluorescence technique to study the behavior of caldesmon during the interaction of myosin subfragment 1 (S1) with thin filaments reconstituted in rabbit skeletal muscle ghost fibers by incorporation of smooth muscle tropomyosin and caldesmon labeled with acrylodan at cysteine residue located in the C-terminal region. Significant changes in acrylodan fluorescence intensity upon addition of skeletal muscle S1 reflected substantial displacement of caldesmon from thin filaments, while alterations in the calculated fluorescence parameters indicated the simultaneous rearrangement of the remaining caldesmon fraction. The orientation of caldesmon in the S1-thin filament complex relative to the fiber axis changes by approximately 7° and the mobility of the fluorescent probe by about 9%. The alterations in caldesmon orientation were proportional to the strength of S1 binding and diminished respectively upon addition of ADP and ADP-V i. The changes in orientation of acrylodan-caldesmon evoked by the interaction of S1 with thin filaments were more pronounced than that in AEDANS-F-actin which suggests that the spatial arrangement of caldesmon in the complex is governed not only by F-actin but also by S1. The results may indicate that the changes in spatial arrangement of caldesmon are adjusted to the conformation of F-actin and S1 characteristic for particular steps of the ATP hydrolysis cycle.

Inhibitory effect of ATP analogs and actin on the modification of myosin subfragment 1 with 9-anthroylnitrile

The fluorescent probe, 9-anthroylnitrile (ANN), can selectively attach to Ser-180 at the ATP-binding site of subfragment 1 (S1) of skeletal muscle myosin [J. Biol. Chem. 278 (2003) 31891]. We have found that MgATP, MgATPγS, MgADP·AlF 4 or MgPP i, but not MgADP, inhibit the incorporation of ANN into S1. The inhibitory effect of the nucleotide γ-phosphate group (or its analog) on the modification of S1 with ANN can be explained by the contribution of Ser-180 to the binding of the nucleotide γ-phosphate at the active site of S1. We have also observed that the incorporation of ANN into S1·MgADP complex is inhibited by actin. These experimental data strongly support the existence of nucleotide-promoted conformational changes revealed by crystal structures of S1 complexes with various nucleotide analogs. They also convincingly show an effect of actin on the environment of Ser-180 at the nucleotide binding site of S1.

Chemical Cross-linking of Actin and Myosin Subfragment-1 in Rigor Complex

Chemical cross-linking of actin and myosin subfragment-1 (S-1) was investigated under various conditions. Actin and S-1 were cross-linked wlth the aid of zero-angstrom cross-linker 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC). Three new bands having larger molecular weight than those of two subunits of S-1 and one new, band having smaller molecular weight than the sub-units of S-1 appeared on electrophoretograms after the cross-linking reaction. These new bands were formed by cross-1inking between actin and subcomponents of S-1. The extent of cross-linking decreased with increasing the ionic strength of reaction mixtures and also decreased with increasing the concentration of ATP. The extent of cross-linking between S-1 and actin prepared from fresh muscle was not appreciably smaller than that of those proteins prepared from postmortem muscle in rigor (stored at O 'C for 24 hr) and even that of those proteins prepared from muscle stored for more prolonged period (168 hr). To elucidate the effect of paratropomyosin on actin-S-1 interaction, the extent of actin-S-1 complex formation in the absence or presence of paratropomyosin during the cross-linking reaction was investigated. As a result, the formation of new bands was reduced after the addition of paratropomyosin, while almost no change of the intensity of S-1 heavy chain (90kDa) was observed. The result indicates that paratropoTnyosin is involved in the weakening of actin-myosin interaction during the development of the resolution of rigor.

Modulation of actomyosin motor function by 1-hexanol.

This study examines the effects of 1-hexanol as a perturbing agent on actomyosin ATPase and its related functions in the concentration range between 0 and 20 mM. In this range the denaturation of myosin subfragment 1 (S1), as measured by the inactivation rate of its K-EDTA-ATPase, and depolymerization of F-actin were insignificant. Major findings showed that hexanol had the following effects which were fully reversible, (a) a marked activation of S1 MgATPase (approximately 10-fold at 20 mM) without greatly affecting the enhancement of tryptophan fluorescence by formation of S1.ADP.Pi intermediate and the rate of ADP release from S1.ADP; (b) an inhibition of the maximum actin-activated ATPase activity; (c) an increase in the affinity of S1 for actin in the presence of ATP and a decrease in the presence of ADP or the absence of nucleotide; (d) a reduction in the sliding velocity of actin filaments in in vitro motility assays with myosin, and (e) a decrease in isometric tension of single skinned muscle fibers. Thus, the effects of hexanol on actomyosin interaction are distinct for the weak and strong binding states, consistent with a change in the hydrophobic interaction in the interface between myosin and actin accompanying the transition from the weak to the strong binding state. Hexanol also accelerates the Pi release from S1.ADP.Pi, which is the transition step from the weak to the strong binding state. The fact that hexanol accelerates Pi release suggests that this alcohol perturbs the S1.ADP.Pi conformation. We speculate that this intermediate-specific structural perturbation is related to the inhibition of the maximum actin-activated ATPase, in vitro motility, and isometric tension.

Multiple Binding of Ligands to a Linear Biopolymer

Publisher Summary Many biological processes involve cooperative binding of several different protein ligands to linear biopolymers (such as DNA, actin, and microtubules). If each subunit (monomer) of the biopolymer has only one ligand-binding site, the binding is purely competitive and cooperative binding phenomena are derived mainly from nearest-neighbor ligand–ligand interactions. This kind of binding is referred to as the ‘‘single-binding’’ type. On the other hand, in ‘‘multiple-binding’’ systems in which each subunit of the biopolymer contains two or more binding sites, such that more than one ligand can bind to the same subunit simultaneously, the cooperative binding of one ligand may be modulated allosterically by the other ligand. For example, binding of myosin subfragment 1 (S-1) molecules to a bare actin is known to be noncooperative. But, when the actin is prebound with tropomyosin–troponin molecules, the binding of S-1 becomes cooperative. Theoretical treatment of cooperative binding of ligands to linear biopolymers is an interesting biophysical problem and has attracted considerable attention. However, most of the formalisms developed so far are concentrated on the ‘‘single-binding’’ model. This chapter introduces a formalism that can be used to treat not only single-binding systems, but also multiple-binding systems. Instead of discussing the theoretical basis of the formalism, this chapter uses a simple two-ligand multiple-binding system to illustrate how to carry out the evaluation of the secular equation and the binding isotherms. The purpose is to present the general methodology of the formalism, so that one may be able to modify or extend when needed.

Effects of tropomyosin internal deletion Delta23Tm on isometric tension and the cross-bridge kinetics in bovine myocardium.

Tropomyosin (Tm) spans seven actin monomers and contains seven quasi-repeating, loosely similar regions, 1-7. Deletion of regions 2-3 decreases the in vitro sliding speed of synthetic filaments of actin-Tm-Troponin (Tn), and weakens Tm binding to the actin-myosin subfragment 1 (S1) complex (acto-S1). The thin filament was selectively removed from bovine myocardium by gelsolin, and the actin filament was reconstituted, followed by further reconstitution with Tm and Tn. In this reconstitution, full-length Tm (control) was compared with Tm internal deletion mutant Delta23Tm, which lacks residues 47-123 (regions 2-3). The effects of phosphate, MgATP, MgADP and Ca2+ were studied in Tm-reconstituted myocardium and Delta23Tm-reconstituted myocardium at pH 7.00 and 25 degrees C. In Delta23Tm, both isometric tension and stiffness were about 40 % of the control. The Hill factor with Delta23Tm, deduced from the pCa-tension plot, was 1.4 times that of the control, but the Ca2+ sensitivity was the same. Sinusoidal analysis indicated that the cross-bridge number in force-generating states was not decreased with Delta23Tm. We conclude that the thin filament cooperativity is increased with Delta23Tm, presumably because of the increased density of the Ca2+-binding sites. We further conclude that tension per cross-bridge is 40 % of control and stiffness per cross-bridge is 40 % of control in Delta23Tm. These results are consistent with the idea that Tm modifies the actin-myosin interface so as to increase the stereospecific interaction between moieties of actin and myosin. In Delta23Tm, the interface may not have a perfect stereospecific match so that the tension- and stiffness-generating capacity is greatly diminished.

C-terminal truncation of cardiac troponin I causes divergent effects on ATPase and force: implications for the pathophysiology of myocardial stunning.

Myocardial stunning is a form of reversible myocardial ischemia/reperfusion injury associated with systolic and diastolic contractile dysfunction. In the isolated rat heart model, myocardial stunning is characterized by specific C-terminal proteolysis of the myofilament protein, troponin I (cTnI) that yields cTnI1-193. To determine the effect of this particular C-terminal truncation of cTnI, without the confounding factor of other stunning-induced protein modifications, a series of solution biochemical assays has been undertaken using the human homologue of mouse/rat cTnI1-193, cTnI1-192. Affinity chromatography and actin sedimentation experiments detected little, or no, difference between the binding of cTnI (cTnI1-209) and cTnI1-192 to actin-tropomyosin, troponin T, or troponin C. Both cTnI and cTnI1-192 inhibit the actin-tropomyosin-activated ATPase activity of myosin subfragment 1 (S1), and this inhibition is released by troponin C in the presence of Ca2+. However, cTnI1-192, when reconstituted as part of the troponin complex (cTn1-192), caused a 54+/-11% increase in the maximum Ca2+-activated actin-tropomyosin-S1 ATPase activity, compared with troponin reconstituted with cTnI (cTn). Furthermore, cTn1-192 increased Ca2+ sensitivity of both the actin-tropomyosin-activated S1 ATPase activity and the Ca2+-dependent sliding velocity of reconstituted thin filaments, in an in vitro motility assay, compared with cTn. In an in vitro force assay, the actin-tropomyosin filaments bearing cTn1-192 developed only 76+/-4% (P<0.001) of the force obtained with filaments composed of reconstituted cTn. We suggest that cTnI proteolysis may contribute to the pathophysiology of myocardial stunning by altering the Ca2+-sensing and chemomechanical properties of the myofilaments.

Cosolvent-induced aggregation inhibits myosin ATPase activity by stabilizing the predominant transition intermediate.

High concentration of the cosolvent poly(ethylene glycol) (PEG) induces reversible aggregation of skeletal myosin subfragment 1 (S1) and inhibition of its Mg-ATPase activity [Highsmith et al. (1998) Biophys. J. 74, 1465-1472]. In the present work the effect of aggregation on the various steps of the ATPase cycle was studied. The isomerization and hydrolysis steps of the cycle were not affected by S1 aggregation since the formation of the "trapped" S1.MgADP.phosphate analogue complexes, which mimic the prehydrolysis M*ATP and posthydrolysis M**ADP.P(i) transition states, proceeded without any hindrance. Similar conclusions could be reached from the chemical modification of Lys-83 and Cys-707 in the presence of MgATP and MgATPgammaS, which indicated that the most populated intermediate of the cycle in solubilized and aggregated S1 is M**ADP.P(i). The dissociation of the trapped S1.MgADP.phosphate analogue complexes resembling the M**ADP.P(i) state was strongly inhibited by PEG-6000, showing that the transition from this intermediate is prevented by the aggregation. This step is presumably inhibited because the coupled swinging of the lever arm from the closed to the open position is constrained by the close packing of aggregated S1.

Visualization of an unstable coiled coil from the scallop myosin rod.

Alpha-helical coiled coils in muscle exemplify simplicity and economy of protein design: small variations in sequence lead to remarkable diversity in cellular functions. Myosin II is the key protein in muscle contraction, and the molecule's two-chain alpha-helical coiled-coil rod region--towards the carboxy terminus of the heavy chain--has unusual structural and dynamic features. The amino-terminal subfragment-2 (S2) domains of the rods can swing out from the thick filament backbone at a hinge in the coiled coil, allowing the two myosin 'heads' and their motor domains to interact with actin and generate tension. Most of the S2 rod appears to be a flexible coiled coil, but studies suggest that the structure at the N-terminal region is unstable, and unwinding or bending of the alpha-helices near the head-rod junction seems necessary for many of myosin's functional properties. Here we show the physical basis of a particularly weak coiled-coil segment by determining the 2.5-A-resolution crystal structure of a leucine-zipper-stabilized fragment of the scallop striated-muscle myosin rod adjacent to the head-rod junction. The N-terminal 14 residues are poorly ordered; the rest of the S2 segment forms a flexible coiled coil with poorly packed core residues. The unusual absence of interhelical salt bridges here exposes apolar core atoms to solvent.