Metal switch-controlled myosin II from Dictyostelium discoideum supports closure of nucleotide pocket during ATP binding coupled to detachment from actin filaments

Abstract

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg(2+)) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg(2+)- or Mn(2+)-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn(2+), providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn(2+) rescues this effect to near wild-type activity. 2'(3')-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.

Keywords: ATPases; Actin; Manganese; Molecular Motors; Myosin.

SCHEME 1

SCHEME 2

FIGURE 1.

Actomyosin cosedimentation experiments. Coomassie Blue-stained SDS gel shows sedimentation of phalloidin-stabilized F-actin with WT and S237C. The supernatant (S) and pellet (P) for each reaction were loaded consecutively with the reaction conditions indicated above each supernatant/pellet pair. Myosin is highlighted with an m and actin with an a. The fractions of myosin in the supernatant and pellet in the presence of nucleotide are labeled at the top of each lane. Final conditions are as follows: 1 μm myosin, 1.5 μm F-actin, 10 mm MgCl₂ or MnCl₂, 10 mm ATP.

FIGURE 2.

Steady-state ATPase showing F-actin concentration dependence. WT (A–C) and S237C (D–F) were reacted with increasing F-actin concentrations with excess MgATP, MnATP, or CaATP (as indicated). Final enzyme concentrations are as follows: A–C and E, 0.25 μm myosin; D and F, 25 nm myosin; 0–70 μm F-actin; 500 μm ATP, 5 mm MgCl₂, MnCl₂, or CaCl₂. A, B, and E were fit to Equation 1, and C, D, and F were fit to Equation 2 (Table 1).

FIGURE 3.

Steady-state ATPase kinetics showing ATP concentration dependence. WT (A–C) and S237C (D–F) were reacted with increasing ATP concentrations and excess MgCl₂, MnCl₂, or CaCl₂ (as indicated) in the absence (closed symbols) or presence (open symbols) of F-actin. Final concentrations are as follows: 0.25 μm myosin, 0 or 10 μm F-actin, 0.25–2400 μm ATP, 5 mm MgCl₂, MnCl₂, or CaCl₂. Each data set was fit to Equation 3 (Table 1).

FIGURE 4.

ATP-promoted dissociation of the actomyosin complex. The complex of pyrenyl-F-actin plus myosin was rapidly mixed with a high ATP concentration and excess of MgCl₂, MnCl₂, or CaCl₂. Final concentrations are as follows: 0.5 μm myosin, 0.25 μm pyrenyl-F-actin, 200 μm ATP, 5 mm MgCl₂, MnCl₂, or CaCl₂. A, control reactions for acto·WT and acto·S237C in the absence of ATP (black and green, respectively) are shown on the short time scale. Other reactions are as indicated. B, longer time scale to observe slow actomyosin dissociation reactions. C, representative transients are shown for ATP-promoted dissociation of the acto·S237C complex at increasing ATP concentrations. Final concentrations are as follows: 0.5 μm S237C, 0.25 μm pyrenyl-F-actin, 0.2–4 mm ATP (as indicated), 5 mm MgCl₂. D, each transient was fit to a single exponential equation, and the observed rate was plotted as a function of ATP concentration. The data were fit to Equation 4, k₊₂ = 0.58 ± 0.06 s⁻¹ and K_{d, ATP} = 617 ± 194 μm.

FIGURE 5.

Kinetics of inorganic phosphate release and ATP binding kinetics for WT and S237C. A, P_i release kinetics by WT (A) and S237C (B) with MgATP (circles) and MnATP (triangles) in the absence of F-actin using the MDCC-PBP-coupled assay. Final concentrations are as follows: 2 μm myosin, 10 μm MDCC-PBP, 0.05 units/ml purine nucleotide phosphorylase, 75 μm 7-methylguanosine, 500 μm ATP, 5 mm MgCl₂ or MnCl₂. Transient for MgATP was fit to a line, k_obs = 0.05 ± 0.0002 s⁻¹ site⁻¹, and the transient for MnATP was fit to Equation 5, k_b = 2.1 ± 0.2 s⁻¹, A_o = 0.22 ± 0.03 μm, and k_ss = 1.0 ± 0.02 s⁻¹ site⁻¹. For S237C in MgATP, k_b = 92 ± 1 s⁻¹, A_o = 0.27 ± 0.001 μm, and k_ss = 0.79 ± 0.003 s⁻¹ site⁻¹. For S237C in MnATP, k_b = 178 ± 13 s⁻¹, A_o = 0.05 ± 0.003 μm, and k_ss = 0.50 ± 0.002 s⁻¹ site⁻¹. Linear (C) and burst (D) fitting of P_i release kinetics for WT reacted with MnATP showing residuals around the fit of the data. E, intrinsic tryptophan fluorescence enhancement for WT and S237C upon binding MgATP or MnATP (as indicated; Table 2). F, WT and S237C myosin tryptophan to mant-ATP FRET in Mg²⁺ and Mn²⁺. Each transient in E and F was fit to a single exponential function.

FIGURE 6.

mantADP release kinetics for WT and S237C. A, preformed myosin·mantADP (1:1) complex in excess MgCl₂ or MnCl₂ (as indicated) was rapidly mixed in a stopped-flow instrument with a high concentration of MgATP or MnATP, respectively, and mant-fluorescence emission was monitored. Final concentrations are as follows: 1 μm myosin, 1 μm mantADP, 1 mm ATP, 5 mm MgCl₂ or MnCl₂. Transients were fit to a single exponential decay. For WT + Mg²⁺, k_obs = 1.24 ± 0.004 s⁻¹ and A_o = 1.44 ± 0.002; WT + Mn²⁺, k_obs = 1.98 ± 0.007 s⁻¹ and A_o = 1.37 ± 0.002; S237C + Mg²⁺, k_obs = 0.90 ± 0.05 s⁻¹ and A_o = 0.06 ± 0.001; S237C + Mn²⁺, k_obs = 0.88 ± 0.003 s⁻¹ and A_o = 1.11 ± 0.001. B, preformed myosin·mantADP (1:1) complex in excess CaCl₂ was rapidly mixed in a stopped-flow instrument with a high concentration of CaATP. Final concentrations are as follows: 1 μm myosin, 1 μm mantADP, 1 mm ATP, 5 mm CaCl₂. Transients were fit to a single exponential decay. For WT + Ca²⁺, k_obs = 31.1 ± 0.1 s⁻¹ and A_o = 0.95 ± 0.002; S237C + Ca²⁺, k_obs = 66.1 ± 0.9 s⁻¹ and A_o = 0.25 ± 0.002. Control reaction shown for the S237C·mantADP complex that was rapidly mixed with CaCl₂ (no ATP).