The myosin C-loop is an allosteric actin contact sensor in actomyosin

Abstract

Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.

Figure 1

SDS–PAGE of WT HMM, mutants G362A and G409V, and chimeras containing skeletal (sCl) and β-cardiac (cCl) C-loop sequences. The myosin heavy chain (MHC) and light chains (RLC and ELC) correspond to molecular masses of 140, 20, and 17 kDa, respectively.

Figure 2

Domain-partitioned S1 heavy chain in the M** conformation. S1 has block peptides designated N-terminal (amino acids 1–144, blue ribbon), U50a (amino acids 145–361, green ribbon), U50b (amino acids 362–462, red tube, top), loop 2 containing (amino acids 526–695, yellow ribbon), converter (amino acids 721–772, red ribbon, bottom), and lever arm (amino acids 773–812, white ribbon). Gear peptides are amino acids 463–525 and 696–720 (black stick backbone bonds).

Figure 3

Free energy change in ΔG (◊), work producing lever arm displacement (□), and back door opening (■) for S1 during a representative M** → M transition. Quantities are rescaled to a unit maximum amplitude facilitating comparison of their shape relative to the time dimension demonstrating the synchronization of events at the active site and lever arm with changes in protein free energy. ΔG decreases during a spontaneous process, indicating the U50a transition presents a large energy barrier to the lever arm swing. Crossing the U50a barrier accompanies product release with the opening of the back door and unlocks the lever arm swinging motion to accomplish the power stroke. Simulation shows that coupling the U50a and U50b conformation transitions removes the free energy barrier to product release. The C-loop is a structured surface loop linking U50a and U50b whose structure is perturbed with actin binding. These circumstances suggest that perturbation of the C-loop with actin binding couples U50a and U50b transitions, causing product release and power stroke initiation.

Figure 4

(A) ATP concentration dependence of k_obs measured for WT (●), G409V (○), and G362A (□). ATP binding to HMM in the absence of actin was assessed using tryptophan fluorescence changes. WT HMM was mixed with ATP, and the changes in the tryptophan fluorescence were recorded and fitted with single exponential giving k_obs. The hyperbola fits to the plots in the form k_obs = (k₋₃ + k₃)[ATP]/([ATP] + l/K₁) are solid lines giving k₋₃ + k₃ and K₁ values quoted in Table 3. (B) Dissociation of ADP from HMM in the absence of actin as measured by tryptophan fluorescence changes in time. HMM was pre-equilibrated with ADP and then mixed with ATP in the stopped-flow system. The exponential fit to the transients gave k_obs. Note that the shorter time scale at the top of the plot corresponds to the G362A data. Experiments with G409V closely resembled those with WT. The k_obs values in these experiments corresponded to rate k₆ in Scheme 3. (C) ADP concentration dependence of k_obs measured for WT (●), G409V (○), and G362A (□) bound to p-F-actin. p-F-actin was equilibrated with WT HMM and then mixed with ATP (100 μM) and ADP (various concentrations) in the stopped-flow system. The exponential fit to the pyrene fluorescence changes transients gave k_obs rates for ATP-induced dissociation of the HMM from actin (Scheme 4). The ADP concentration dependence of k_obs reflects the affinity of ADP for the actomyosin, giving K_AD (Table 3). k₀ is k_obs at 0 mM ADP.

Figure 5

In vitro motility histograms (■) and their decomposition into group 1 Gaussian distributions (—) for WT, cCl, and sCl HMM. Rh-phalloidin-labeled bare actin (A) or that with bound human cardiac tropomyosin (HCTm-A) glides over the surface-bound HMM. The fitted black curves are the sums of dashed curves. The left (right) column corresponds to low (high) myosin surface densities.

Scheme 1

Scheme 2

Scheme 3

Scheme 4