Catalytic strategy used by the myosin motor to hydrolyze ATP

Abstract

Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (P i ) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantumclassical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HP γ O 4 2− state, but in the H 2 P γ O 4 − state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (P γ O 3 − ) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.T he molecular motor myosin cyclically interacts with the actin filament to generate the mechanical force that is used in living cells to achieve muscle contraction (1), cytokinesis (2, 3), and intracellular cargo transport (4). Hydrolysis of one ATP molecule per cycle provides the free energy that drives the actomyosin interaction cycle, as originally described by Lymn and Taylor (5). ATP is the common energy currency in biology, and is extremely stable in aqueous solution (6, 7). ATPases, the enzymes that catalyze the hydrolysis of the P β -O-P γ anhydride linkage in ATP, are ubiquitous in biology because they are needed to accelerate the release of free energy stored in ATP. Myosin manages to speed up the hydrolysis by a factor of 10 7 over the uncatalyzed rate in solution. The experimental uncatalyzed energy barrier is 29 kcal mol −1 (7,8), and the catalyzed barrier has been determined experimentally between 14.4 kcal mol −1 (9) and 14.8 kcal mol −1 (10). Understanding how myosin achieves its ATPase function is necessary to understand how myosin works as a motor, but also helps one to understand the functioning of the multitude of other nucleotide hydrolyzing enzymes. The catalytic mechanism of ATP hydrolysis in myosin has been studied extensively with methods such as protein crystallography (11)(12)(13)(14), mutagenesis (15, 16), photochemical kinet...

Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors

“…In all pathways that were recently found for myosin in two independent groups by calculating minimum energy paths [44,48], the dissociation of the γ-phosphate of ATP 4-(into ADP 3-and metaphosphate P γ O 3 -) precedes the deprotonation of the lytic water molecule W a (Figure 5a 5b). The transition state of that process (TS2 diss in Figure 1B) was found to have a potential energy barrier relative to the reactant state (bound ATP, R in Figure 1) of 8.1 kcal mol -1, [44] to 8.7 kcal mol -1 [44], consistent with the experimental enthalpy barrier of 10.3 kcal mol -1 obtained from an Arrhenius plot between 20 and 50 ºC [68].…”

“…In this section, the most recent catalytic pathways that have been computed for ATP hydrolysis in myosin [48,67], kinesin [43] and F 1 -ATPase [32] are summarized. It turns out that the chemical strategy of all three motors is essentially identical and follows the scenario shown at the bottom of Figure 1B (i.e., via M2).…”

“…After crossing TS2 diss , the resulting metaphosphate intermediate (M2 in Figure 1B) has been found to be a low potential energy minimum, only 2.6±0.2 kcal mol -1 above the reactant state R [40,48]. In the M2 state, the metaphosphate forms a hydrate complex with water W a (H 2 O a ·P γ O 3 -), so that the distance between O a and P γ is very short (1.9 Å) [40].…”

“…[20][21][22][23][24][25][26][27][28] This is because a) experimental techniques cannot convincingly differentiate between the sequential or concurrent nature [29] of the hydrolysis mechanism ( Figure 1), [30,31] b) mutagenesis experiments can introduce structural perturbations in the active site of mutated enzymes, [32] and c) it is difficult to distinguish the effects from conformational changes during ligand binding/unbinding onto the observed k cat . [33] Recently, advances in combined quantum/classical (QM/MM) simulations [20,[34][35][36][37][38] have allowed the exploration of the reaction of ATP hydrolysis in ATP-driven biomolecular motors at atomic detail [32,[39][40][41][42][43][44][45][46][47][48].…”

Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction

“…In the cytoplasm of cardiomyocyte, actin-based myosin motors are responsible for muscle contraction in an ATP-dependent manner [8,13]. Although the contractile function of cytoplasmic myosin has been well investigated, the roles of nuclear myosins have only attracted attention in recent years.…”