Ewa Prochniewicz

Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament

We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate and (b) copolymerization of unmodified actin monomers with monomers cross-linked internally with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Each of these modifications abolished the interaction of the modified monomers with myosin, so the remaining interactions were exclusively with unmodified monomers. The two modifications had similar effects on the interaction of actin with myosin in solution: decreased affinity of myosin heads for unmodified actin monomers, without a change in the Vmax of actin-activated myosin ATPase activity. However, modification (b) produced much greater inhibition of actin sliding on a myosin-coated surface, as measured by an in vitro motility assay. These results provide insight into the functional consequences of cooperative interactions within the actin filament.

Dystrophin and utrophin have distinct effects on the structural dynamics of actin

We have used time-resolved spectroscopy to investigate the structural dynamics of actin interaction with dystrophin and utrophin in relationship to the pathology of muscular dystrophy. Dystrophin and utrophin bind actin in vitro with similar affinities, but the molecular contacts of these two proteins with actin are different. It has been hypothesized that the presence of two low-affinity actin-binding sites in dystrophin allows more elastic response of the actin–dystrophin–sarcolemma linkage to muscle stretches, compared with utrophin, which binds via one contiguous actin-binding domain. We have directly tested this hypothesis by determining the effects of dystrophin and utrophin on the microsecond rotational dynamics of a phosphorescent dye attached to C374 on actin, as detected by transient phosphorescence anisotropy (TPA). Binding of dystrophin or utrophin to actin resulted in significant changes in the TPA decay, increasing the final anisotropy (restricting the rotational amplitude) and decreasing the rotational correlation times (increasing the rotational rates and the torsional flexibility). This paradoxical combination of effects on actin dynamics (decreased amplitude but increased rate) has not been observed for other actin-binding proteins. Thus, when dystrophin or utrophin binds, actin becomes less like cast iron (strong but brittle) and more like steel (stronger and more resilient). At low levels of saturation, the binding of dystrophin and utrophin has similar effects, but at higher levels, utrophin caused much greater restrictions in amplitude and increases in rate. The effects of dystrophin and utrophin on actin dynamics provide molecular insight into the pathology of muscular dystrophy.

Myosin Isoform Determines the Conformational Dynamics and Cooperativity of Actin Filaments in the Strongly Bound Actomyosin Complex

We used transient phosphorescence anisotropy to detect the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin strongly bound to single-headed fragments of muscle myosin subfragment 1 (S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at substoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than with muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects: muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly and muscle myosin binds more slowly to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound erythrosin iodoacetamide: while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex are determined by the isoform of the bound myosin in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells.

Age-related decline in actomyosin structure and function

This review focuses on the role of changes in the contractile proteins actin and myosin in age-related deterioration of skeletal muscle function. Functional and structural changes in contractile proteins have been determined indirectly from specific force and unloaded shortening velocity of permeabilized muscle fibers, and were detected directly from site-directed spectroscopy in muscle fibers and from biochemical analysis of purified actin and myosin. Contractile proteins from aged and young muscle differ in (a) myosin and actomyosin ATPase activities, (b) structural states of myosin in contracting muscle, (c) the state of oxidative modifications. The extent of age-related physiological and molecular changes is dependent on the studied animal, the animals age, and the type of muscle. Therefore, understanding the aging process requires systematic, multidisciplinary studies on physiological, biochemical, structural, and chemical changes in specific muscles.

Large-scale opening of utrophin's tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin–actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron–electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.

Changes in actin structural transitions associated with oxidative inhibition of muscle contraction.

We have used transient phosphorescence anisotropy (TPA) to detect changes in actin structural dynamics associated with oxidative inhibition of muscle contraction. Contractility of skinned rabbit psoas muscle fibers was inhibited by treatment with 50 mM H 2O 2, which induced oxidative modifications in the myosin head and in actin, as previously reported. Using proteins purified from oxidized and unoxidized muscle, we used TPA to measure the effects of weakly (+ATP) and strongly (no ATP) bound myosin heads (S1) on the microsecond dynamics of actin labeled at Cys374 with erythrosine iodoacetamide. Oxidative modification of S1 had no effect on actin dynamics in the absence of ATP (strong binding complex), but restricted the dynamics in the presence of ATP (weakly bound complex). In contrast, oxidative modification of actin did not have a significant effect on the weak-to-strong transitions. Thus, we concluded that (1) the effects of oxidation on the dynamics of actin in the actomyosin complex are predominantly determined by oxidation-induced changes in S1, and (2) changes in weak-to-strong structural transitions in actin and myosin are coupled to each other and are associated with oxidative inhibition of muscle contractility.

Changes in actin and myosin structural dynamics due to their weak and strong interactions.

Figure 3 summarizes the effects of actomyosin binding on the internal and global dynamics of either protein, as discussed in this chapter. These effects depend primarily on the strength of the interaction; which in turn depends on the state of the nucleotide at the myosin active site. When either no nucleotide or ADP is bound, the interaction is strong and the effect on each protein is maximal. When the nucleotide is ATP or ADP.Pi, or the equivalent nonhydrolyzable analogs, the interaction is weak and the effect on molecular dynamics of each protein is minimal. The weaker effects in weak-binding states are not simply the reflection of lower occupancy of binding sites--the molecular models in Fig. 3 illustrate the effects of the formation of the ternary complex, after correction for the free actin and myosin in the system. Thus EPR on myosin (Berger and Thomas 1991; Thomas et al. 1995) and pyrene fluorescence studies on actin (Geeves 1991) have shown that the formation of a ternary complex has a negligible effect on the internal dynamics of both [figure: see text] proteins (left side of Fig. 3, white arrows). As shown by both EPR (Baker et al. 1998; Roopnarine et al. 1998) and phosphorescence (Ramachandran and Thomas 1999), both domains of myosin are dynamically disordered in weak-binding states, and this is essentially unaffected by the formation of the ternary complex (left side of Fig. 3, indicated by disordered myosin domains). The only substantial effect of the formation of the weak interaction that has been reported is the EPR-detected (Ostap and Thomas 1991) restriction of the global dynamics of actin upon weak myosin binding (left column of Fig. 3, gray arrow). The effects of strong actomyosin formation are much more dramatic. While substantial rotational dynamics, both internal and global, exist in both myosin and actin in the presence of ADP or the absence of nucleotides, spin label EPR, pyrene fluorescence, and phosphorescence all show dramatic restrictions in these motions upon formation of the strong ternary complex (right column of Fig. 3). One implication of this is that the weak-to-strong transition is accompanied by a disorder-to-order transition in both actin and myosin, and this is itself an excellent candidate for the structural change that produces force (Thomas et al. 1995). Another clear implication is that the crystal structures obtained for isolated myosin and actin are not likely to be reliable representations of structures that exist in ternary complexes of these proteins (Rayment et al. 1993a and 1993b; Dominguez et al. 1998; Houdusse et al. 1999). This is clearly true of the strong-binding states, since the spectroscopic studies indicate consistently that substantial changes occur in both proteins upon strong complex formation. For the weak complexes, the problem is not that complex formation induces large structural changes, but that the structures themselves are dynamically disordered. This is probably why so many different structures have been obtained for myosin S1 with nucleotides bound--each crystal is selecting one of the many different substates represented by the dynamic ensemble. Finally, there is the problem that the structures of actomyosin complexes are probably influenced strongly by their mechanical coupling to muscle protein lattice (Baker at al. 2000). Thus, even if co-crystals of actin and myosin are obtained in the future, an accurate description of the structural changes involved in force generation will require further experiments using site-directed spectroscopic probes of both actin and myosin, in order to detect the structural dynamics of these ternary complexes under physiological conditions.

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15–20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5–C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0–C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin–myosin interaction.

Binding of Dystrophin’s Tandem Calponin Homology Domain to F-Actin Is Modulated by Actin’s Structure

Dystrophin has been shown to be associated in cells with actin bundles. Dys-246, an N-terminal recombinant protein encoding the first 246 residues of dystrophin, includes two calponin-homology (CH) domains, and is similar to a large class of F-actin cross-linking proteins including alpha-actinin, fimbrin, and spectrin. It has been shown that expression or microinjection of amino-terminal fragments of dystrophin or the closely related utrophin resulted in the localization of these protein domains to actin bundles. However, in vitro studies have failed to detect any bundling of actin by either intact dystrophin or Dys-246. We show here that the structure of F-actin can be modulated so that there are two modes of Dys-246 binding, from bundling actin filaments to only binding to single filaments. The changes in F-actin structure that allow Dys-246 to bundle filaments are induced by covalent modification of Cys-374, proteolytic cleavage of F-actins C-terminus, mutation of yeast actins N-terminus, and different buffers. The present results suggest that F-actins structural state can have a large influence on the nature of actins interaction with other proteins, and these different states need to be considered when conducting in vitro assays.

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

Significance Myosin is an enzyme that uses energy from ATP to exert force on another protein, actin, resulting in muscle contraction. Force is generated in the power stroke, when the actin–myosin complex transitions from a weak-binding structure (ATP bound to myosin) to a strong-binding structure (ADP). We detected the amplitude of the power stroke directly by measuring fluorescence resonance energy transfer from actin to myosin, with high time resolution following excitation with a pulsed laser. We found that the amplitude of the power stroke is strongly dependent on the form of the essential light chain that is bound to myosin. This provides a structural explanation for previous functional observations, with implications for muscle pathophysiology and therapeutic design. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.