Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding

Abstract

Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the SH1-SH2 helix of the myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the SH1-SH2 helix modulate the affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C₂C₁₂ cells, and the duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. The mutant R694N showed a faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus indicating that electrostatic interactions within the SH1-SH2 helix region of human cardiac myosin modulate ADP release and thus, the duration of the strongly bound state of actomyosin.

Keywords: ADP; ATP; Actin; Electrostatics; Myosin; Salt bridge; Transient kinetics.

Figure 1.

The SH1-SH2 helix of the myosin head. Left panel, alpha isoform, right panel – beta isoform. Spheres – charged residues, sticks – permanent salt bridges. The bridge E700:R703 exists in both isoforms, the bridge E98:R703 in beta isoform switches to the bridge E98:R708 in alpha isoform. Bridges E98:R694 and E45:R694 exist only in the beta isoform.SH1 helix unwinds in beta isoform in the MD simulations [6].

Figure 2.

SDS-PAGE of the purified recombinant myosin head. 98 kDa human cardiac S1 co-purifies with murine ELC and RLC.

Figure 3.

Typical transients in the experiment of the ATP-induced actomyosin dissociation with and without ADP. Actomyosin (0.5 μM) rapidly mixed with ATP (upper trace, black), or the mixture of ATP and ADP (lower trace, blue). [ATP] = 900 μM in both cases. [ADP] = 200 μM when present in the mixture. Final pyrene fluorescence is the same, indicating complete actomyosin dissociation regardless of the concentration of ADP used. Fitting curves (red) are single exponential function (upper trace, the actomyosin + ATP experiment) and double exponential function (lower trace, the actomyosin + (ATP+ADP) experiment). Deadtime, measured in a separate experiment, constrains the fit (all fits intercept at the mixing time in the bottom left corner). The vertical dashed line shows the time of the flow stop and the beginning of the fit. Meaningful kinetic traces lay on the right side of the dashed line. On the left, there are flow artifacts.

Figure 4.

Rate of ATP-induced actomyosin dissociation. Black circles, WT, N=3, red squares, R694N mutant, data from [6], N=3 (the point at [ATP]=600 μM, N=1), blue triangles, E45Q mutant, N=3. Reaction rates fitted with a hyperbola, k_+2T = 491.5±74.1 s⁻¹, 338.8±47.8 s⁻¹, 618.5±75.3 s⁻¹ for WT, R694N, and E45Q respectively. Data points are mean ± SD. N is the number of biological replicates.

Figure 5.

ATP-induced actomyosin dissociation. Observed reaction rates at low [ATP] are fitted by a straight line, the second-order reaction rate constant is determined from the slope of the line. Black circles, WT, N=3, and red squares R694N mutant, N=3, data from [6]. Blue triangles, E45Q mutant, N=3. K_1TK_+2T = 2.12±0.21 μM⁻¹s⁻¹, 1.85±0.03 μM⁻¹s⁻¹, 3.27±0.31 μM⁻¹s⁻¹ for WT, R694N, and E45Q respectively. Data points are mean ± SD. N is the number of biological replicates.

Figure 6.

Typical transients in the ATP-induced actomyosin dissociation experiment. Actomyosin (0.5 μM) is rapidly mixed with ATP (900 μM, upper trace, black dots, or 60 μM, lower trace, blue dots). The transients are fitted to the numerical solution of differential equations (Eq. S1), red curves. All transients obtained for the same myosin preparation are fitted globally with the same set of kinetic constants. A, WT myosin, B, R694N myosin mutant, C, E45Q myosin mutant.

Figure 7.

Typical transients in the experiment when actomyosin (0.5 μM) is rapidly mixed with the premixed ATP and ADP. [ATP] = 900 μM (WT and R694N) and 600 μM (E45Q). [ADP] = 0 μM, upper trace, black dots, and [ADP] = 100 μM, lower trace, blue dots. The transients are fitted to the numerical solution of differential equations (Eq. S2), red curves. All transients obtained for the same myosin preparation are fitted globally with the same set of kinetic constants. A, WT myosin, B, R694N myosin mutant, C, E45Q myosin mutant.

Scheme 1.

ATP-induced actomyosin dissociation. A = pyrene-labeled actin, M = myosin, T = ATP. A* = actin with unquenched pyrene fluorescence.

Scheme 2.

ATP-induced actomyosin dissociation, competitive inhibition with ADP.

Scheme 3.

ATP-induced actomyosin dissociation, inhibition with ADP. Two sequential states of the actomyosin ADP complex considered. Superscript⁽²⁾ indicates rate constants of the two-step reaction mechanism.