Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Abstract

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times Muscle contraction involves the sliding of actin filaments relative to myosin filaments (1, 2), which is driven by cyclic interactions of the myosin head domains with the actin filaments, powered by hydrolysis of ATP. During each ATP-hydrolysis cycle chemical energy of ATP hydrolysis is transformed into mechanical work by a multistep power-stroke which drives the actin filaments several nanometers past the myosin filaments. The power-stroke is associated with the release of the ATP hydrolysis products, P i (inorganic phosphate) and ADP, from the active site (3). It is generally believed that P i release from the AM⅐ADP⅐P i 3 complex is closely related to the initiation of the power-stroke (4) and to the transition of the myosin head domain from states of weak and non-stereospecific actin binding to states of strong and stereospecific binding to actin (5-7). Because of the assumed close relation between power stroke and the release of inorganic phosphate from the myosin head domain, studying the effects of inorganic phosphate on contracting muscle fibers became a main element to elucidate the relation between P i release and force generation. Effects of P i were examined on isometric force (4, 8 -14) and unloaded shortening velocity (9, 13, 15) as well as on force transients in response to release of P i from caged P i (16,17). As a result of these studies it was proposed that inhibition of active force by inorganic phosphate results from rebinding of P i to the AM⅐ADP intermediate that is formed after P i release (cf. Scheme 1). Thus, the release of phosphate is reversed and a strong binding force generating cross-bridges in the AM⅐ADP state are reversed to a weak binding non-force generating AM⅐ADP⅐P i state (the AM⅐ADP⅐Pi I state in Scheme 1) that is in rapid equilibrium with the detached M⅐ADP⅐P i I intermediate. To fully account for the observed steady state kinetic data and the force transients recorded upon release of P i from caged P i , it was proposed that a strong binding AM⅐ADP⅐P i intermediate (the AM⅐AD...

Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

“…As expected, elevating P i depressed V actin at subsaturating ATP, consistent with previous observations (1,15,37). This effect, which was strongest at pH 7.4, was still present, albeit smaller in magnitude, at pH 6.8 and was completely reversed at pH 6.5 (Fig.…”

“…This result suggests that elevated levels of [P i ] significantly depress the rate of k ϩATP of myosin. This is consistent with the idea that P i readily competes with ATP for binding to myosin's active site (1,27).…”

“…In agreement with the findings in muscle fibers (27), P i was found to cause a mild increase in actin filament velocity (V actin ) at saturating ATP (14). Also in agreement with observations in muscle fibers, several authors reported that elevated levels of P i decrease V actin at micromolar levels of ATP in the in vitro motility assay (1,15,37).…”

“…In agreement with the findings in muscle fibers (27), P i was found to cause a mild increase in actin filament velocity (V actin ) at saturating ATP (14). Also in agreement with observations in muscle fibers, several authors reported that elevated levels of P i decrease V actin at micromolar levels of ATP in the in vitro motility assay (1,15,37).Since the P i -induced increases in velocity have typically been small and not consistently observed, researchers have focused on identifying the molecular basis of the depressive effects of P i on V actin . Pate and Cooke (27) were the first to suggest that this effect was explained by P i competing with ATP for the active site of myosin.…”

“…In addition, we also assumed 0.025% contaminating pyrophosphate (inorganic pyrophosphate), which has a higher affinity for myosin than P i (1). However, recent evidence indicates that this amount of inorganic pyrophosphate contamination has no discernable effect on myosin function in an in vitro motility assay (1).…”