Holding two heads together: stability of the myosin II rod measured by resonance energy transfer between the heads

Abstract

Myosin, similar to many molecular motors, is a two-headed dimer held together by a coiled-coiled rod. The stability of the coiled coil has implications for head-head interactions, force generation, and possibly regulation. Here we used two different resonance energy transfer techniques to measure the distances between probes placed in the regulatory light chain of each head of a skeletal heavy meromyosin, near the head-rod junction (positions 2, 73, and 94). Our results indicate that the rod largely does not uncoil when myosin is free in solution, and at least beyond the first heptad, the subfragment 2 rod remains relatively intact even under the relatively large strain of two-headed myosin (rigor) binding to actin. We infer that uncoiling of the rod likely does not play a role in myosin II motility. To keep the head-rod junction intact, a distortion must occur within the myosin heads. This distortion may lead to different orientations of the light-chain domains within the myosin dimer when both heads are attached to actin, which would explain previously puzzling observations and require reinterpretation of others. In addition, by comparing resonance energy transfer techniques sensitive to different dynamical time scales, we find that the N terminus of the regulatory light chain is highly flexible, with possible implications for regulation. An intact rod may be a general property of molecular motors, because a similar conclusion has been reached recently for kinesin, although whether the rod remains intact will depend on the relative stiffness of the coiled coil and the head in different motors.

Figure 1

Model of two-headed myosin binding to actin and position of probes with the LC domains undistorted, implying an uncoiling of S2 (a), or distorted to fit our observed distances, implying a largely coiled S2 (b). Two S1 myosin heads were docked to F-actin (gray) according to the model of Rayment, Holmes, and coworkers (23) with each head following an undistorted actin helix, leading to distances of ≈90 Å (a). With the catalytic regions remaining rigidly in this conformation, the LC domain of the leading (Lower) head was moved to decrease the distance to the trailing (Upper) LC domain, the position of which was not changed. The orientation of the myosin LC domain was rotated about amino acid 770 (yellow; ref. 31), and distances were measured between the two RLCs, residues 94 to 94 or 73 to 73. In addition, the distance between the two C-terminal ends of the heavy chains, i.e., amino acid 843, was measured also to ascertain the degree of unfolding required in S2. When the bottom head was distorted by rotating its LC domain by 25° from the actin axis (axial angle) and a change in the azimuthal angle by 15°, the three distances all decreased to ≈50 Å (b). The myosin heavy chains are shown in red. The essential LCs (cyan) and RLCs (blue) are shown, as are the distances between Cys-94s (pink), Cys-73s (green), and residues 843 (black), where S2 begins. Cys-2 of the RLC is not visible in the crystal structure. (Inset) The structure of the lanthanide chelate donor, terbium diethylenetriaminepentacetate-carbostyril 124-maleimidopropionic hydrazide (Tb-DTPA-cs124-EMPH), is shown.

Figure 2

(a) Steady-state FRET on probes at Cys-73 with and without actin. Donor-acceptor (D-A) emission includes donor emission and acceptor emission arising from direct excitation and sensitized emission. Subtracting off the direct acceptor emission leaves donor emission plus sensitized emission of acceptor (D-A sens. emission). Significant sensitized emission is evident, indicating energy transfer and relatively close proximity between a donor on one head and an acceptor on the other head of HMM. For this data set, distances are 49.3 and 48.7 Å with and without actin, respectively. For more details see text. Black curves, HMM with actin; gray curves, HMM without actin. (b) Lifetime FRET on the Cys-73 mutant. The data shown are without actin. Lifetimes with actin are very similar (data not shown for clarity of presentation). Donor (D) only = 100% 4.02 nsec, χ² = 0.2 (4.0 nsec with actin). Donor-acceptor (D-A) without actin fit to 20% donor only + 52% 1.59 nsec + 28% 0.36 nsec, χ² = 0.6: average E = 72% for those donors with acceptors, corresponding to R = 47 Å. With actin, donor-acceptor fit to 21% donor only + 79% 0.98 nsec (χ² = 3.3), or E = 76%, R = 46 Å. These results indicate the probes at Cys-73 remain in close proximity when HMM is free or bound to actin. (c) Lifetime LRET on the Cys-73 mutant. The data are shown for both with and without actin, although in all cases decays with and without actin were very similar and showed significant energy transfer. The donor-only curve (black) with and without actin was indistinguishable and fit to a two-exponential decay: τ_d = 38%exp(−t/725) + 62%exp(−t/1823), χ² = 1.03, 〈τ_d〉 = 1,401 μsec. Donor-acceptor (DA) emissions are measured at the terbium emission peak at 546 nm (pink curve is without actin; blue curve is with actin). For the donor-acceptor fit at 545 nm (without actin), we have 21% donor only + 41% 217 μsec + 39% 963 μsec (χ² = 1.06, 〈τ_da〉 = 581 μsec), where the latter two time constants correspond to τ_da, which implies that 80% of the donors are paired with acceptors, and these donors undergo an 〈E〉 of 59%, corresponding to a donor-acceptor distance of 53 Å. For the donor-acceptor fit with actin we have 22% donor only + 40% 205 μsec + 37% 894 μsec (χ² = 0.97, 〈τ_da〉 = 536 μsec). E calculated from 〈τ_da〉 equals 62%, corresponding to a distance of 52 Å. Donor-acceptor-sensitized emission measured at 570 nm (τ_ad) without actin (red curve) fit to 2.4% donor only (because of donor leakage into sensitized emission channel) + 79% 64 μsec + 19% 338 μsec, χ² = 1.48. Converting the amplitudes into populations (12) yields a 〈τ_ad〉 = 232 μsec and 〈E〉 = 83.4%, corresponding to an average distance of 43 Å. With actin, the donor-acceptor-sensitized emission measured at 570 nm (green curve) fit to 2.4% donor only + 80% 63 μsec + 18% 343 μsec, χ² = 1.40; a 〈τ_ad〉 = 231.5 μsec, 〈E〉 = 83.5%, corresponding to an average distance of 43 Å. The multiexponential nature of the decays is discussed further in the text.

Figure 3

Steady-state FRET (a) and lifetime LRET (b) on Cys-2. Steady-state FRET shows almost no sensitized emission, indicating probes are on average far apart. LRET measurements show significant donor-lifetime shortening and sensitized emission; hence probes can come close on the submillisecond time scale. For b, the same color scheme as that described for Fig. 2c is used. From donor-acceptor (D-A) emission, the average distance is 53 Å with actin and 52 Å without actin. From sensitized emission, the distances are 44 Å with actin and 45 Å without actin.