Structural Dynamics of Protein Interactions Using Site-Directed Spin Labeling of Cysteines to Measure Distances and Rotational Dynamics with EPR Spectroscopy.

Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, ß-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA's function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.

Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.

A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure.

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.

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.