Piyali Guhathakurta

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.

The structural dynamics of actin during active interaction with myosin depends on the isoform of the essential light chain.

We have used time-resolved phosphorescence anisotropy to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin concentrations but substantially higher apparent actin affinity, resulting in a higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actins final anisotropy cooperatively and to a similar extent, indicating a similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of a saturating level of ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin.

Allosteric communication in Dictyostelium myosin II

Myosin’s affinities for nucleotides and actin are reciprocal. Actin-binding substantially reduces the affinity of ATP for myosin, but the effect of actin on myosin’s ADP affinity is quite variable among myosin isoforms, serving as the principal mechanism for tuning the actomyosin system to specific physiological purposes. To understand the structural basis of this variable relationship between actin and ADP binding, we studied several constructs of the catalytic domain of Dictyostelium myosin II, varying their length (from the N-terminal origin) and cysteine content. The constructs varied considerably in their actin-activated ATPase activity and in the effect of actin on ADP affinity. Actin had no significant effect on ADP affinity for a single-cysteine catalytic domain construct, a double-cysteine construct partially restored the actin-dependence of ADP binding, and restoration of all native Cys restored it further, but full restoration of function (similar to that of skeletal muscle myosin II) was obtained only by adding all native Cys and an artificial lever arm extension. Pyrene-actin fluorescence confirmed these effects on ADP binding to actomyosin. We conclude that myosin’s Cys content and lever arm both allosterically modulate the reciprocal affinities of myosin for ADP and actin, a key determinant of the biological functions of myosin isoforms.

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin–myosin structure and function. Actin–myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin–myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosins essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin–ANT complex as in the actin–myosin complex and (b) a significant decrease in actin–ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these “hits” alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin–ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin–myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

Actin-Myosin Interaction: Structure, Function and Drug Discovery

Actin-myosin interactions play crucial roles in the generation of cellular force and movement. The molecular mechanism involves structural transitions at the interface between actin and myosin’s catalytic domain, and within myosin’s light chain domain, which contains binding sites for essential (ELC) and regulatory light chains (RLC). High-resolution crystal structures of isolated actin and myosin, along with cryo-electron micrographs of actin-myosin complexes, have been used to construct detailed structural models for actin-myosin interactions. However, these methods are limited by disorder, particularly within the light chain domain, and they do not capture the dynamics within this complex under physiological conditions in solution. Here we highlight the contributions of site-directed fluorescent probes and time-resolved fluorescence resonance energy transfer (TR-FRET) in understanding the structural dynamics of the actin-myosin complex in solution. A donor fluorescent probe on actin and an acceptor fluorescent probe on myosin, together with high performance TR-FRET, directly resolves structural states in the bound actin-myosin complex during its interaction with adenosine triphosphate (ATP). Results from these studies have profound implications for understanding the contractile function of actomyosin and establish the feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

Cardiomyopathy Mutation in Ventricular Essential Light Chain of Cardiac Myosin Alters Structural and Functional Interaction with Actin

We have used site-directed time-resolved FRET (TR-FRET) to determine the effect of pathological mutations in human ventricular essential light chain (hVELC) of cardiac myosin on interaction with actin. The hVELC modulates function of actomyosin, and the proposed mechanisms involve interaction of its N-terminus with actin and its C-terminal region with the IQ domain of myosin. Several mutations in hVELC are associated with familial hypertrophic cardiomyopathy (FHC), which disturbs morphology of the heart and can lead to sudden death. We focus on the cardiomyopathy mutation E56G, which was shown to affect binding of hVELC to the IQ domain. We have introduced this mutation in a single cysteine hVELC construct (A16C) and exchanged both ELCs (A16C and A16C-E56G) onto bovine cardiac S1. This mutation altered actin-activated S1 ATPase by increasing both Vmax and KATPase, suggesting different structural transitions from the weakly to strongly bound states in the ATPase cycle. Using a donor fluorescent probe IAEDANS on actin (at C374) and an acceptor probe Dabcyl maleimide on the N-lobe (A16C) of hVELC, we used TR-FRET to measure the distance between probes on actin and ELC for both proteins. Our previous studies showed that this distance is a sensitive measure of the weak-to-strong transition in the actomyosin complex. Current results indicate that E56G has no significant effect on the structure of strongly bound actomyosin, but fundamentally changes distribution of structural states in the presence of ATP. We conclude that the primary pathogenic effect of the E56G mutation is on the distribution of actomyosin structural states, affecting contractility and remodeling of cardiac muscle, with disastrous results.

Effect of Actin and Nucleotide on the Movement of A1-Type Myosin Essential Light Chain, Detected by Time-Resolved FRET

We have used site-directed time-resolved FRET to detect structural changes of the N-terminal extension on the A1 isoform of ELC, during active interaction with actin. Skeletal muscle myosin subfragment 1 (S1) has two ELC isoforms, A1 and A2, which differ by the presence of 40-45 additional residues at the N-terminus of A1. Removal of ELC from myosin results in a loss of movement of actin filaments, and a reduction in isometric force. It has been proposed that the N-terminal extension of A1 interacts directly with actin to modulate actomyosin kinetics. We have used time-resolved FRET to explore the structural details of this modulation. We recently showed, using probes on actin and ELC, that the amplitude of the myosin power stroke is much greater for A1 than A2. We have now engineered single cysteine (C16, in the N-terminal extension) and double cysteine (C16-C180, in the C-terminal lobe) mutants of A1-ELC and used FRET to determine the distance from C16 to actin 374 or to C180, as affected by the power stroke. Labeled ELCs were exchanged into S1, and the pure isoenzyme (S1A1) was isolated using Talon affinity resin. Labeling and exchange preserved the functional properties of S1A1. Intermolecular FRET between C374 of actin and C16 of A1 showed that the distance increases from 2.9 nm to 3.5 nm upon addition of ATP. Intramolecular FRET between C16 and C180 does not change in the presence of ATP or actin. We conclude that (1) the N-terminus of ELC moves away from actin during the transition between strongly and weakly bound states of acto-S1, and (2) the converter/C-terminal domain of myosin/ELC and the N-terminal extension move as a rigid body during the power stroke.

Allosteric Effects within the Catalytic Domain of Dictyostelium Myosin on Interaction with Actin and Nucleotide

To understand molecular features of myosin critical for its functional interaction with actin, we studied two constructs of Dictyostelium myosin II, which differ in cysteine content and length: (i) Cys-lite catalytic domain (M758-S619C) and (ii) fusion of wild type catalytic domain with two α-actinin repeats (M761-2R), which has been shown to be kinetically identical to full-length Dictyostelium myosin S1. In the absence of ATP, both constructs bound actin stoichiometrically. However, marked differences in interaction of each construct with actin were observed in the presence of ATP. Actin-activated ATPase of M758-S619C was very sensitive to the presence of an ATP regenerating system (i.e., elimination of free ADP), which increased activity by a factor of 2.5, while M761-2R was not affected. The ATP regenerating system greatly decreased actin binding of M758-S619C, as measured by cosedimentation. Fluorescence resonance energy transfer between IAEDANS-actin and IAF-M758-S619C showed that this interaction was strong in the presence of ATP, independent of ionic strength, and structurally indistinguishable from that of the strong-binding complex. These results indicated that M758-S619C has an altered relationship between the binding of actin and nucleotide. Binding of MantADP confirmed this hypothesis: actin had no significant effect on the tight binding (Kd ∼ 1 μM) of MantADP to M758-S619C, while actin substantially decreased the affinity of MantADP for M761-2R, as for muscle S1. We conclude that the structural alterations of the M758-S619C construct (Cys content and/or the structure of the converter domain) allosterically modulates the reciprocal affinity of myosin for nucleotide and actin, which in turn changes the distribution of actomyosin states within the actomyosin ATPase cycle.