Myosin 5A Walking Mechanism: The Structural Basis of Slow ADP Dissociatin from the Lead Head

Abstract

Using electron microscopy and image averaging, we have observed myosin 5a walking along actin filaments in the presence of low concentrations of ATP. Most molecules are attached with 13 actin subunits between heads but ∼10% are bound with 11 or 15 subunit spacings. Most lead heads are in the pre-powerstroke conformation, but some post-powerstroke lead heads are observed, especially at smaller separations where there is less strain in the myosin. Post-powerstroke lead heads have the converter at the front of the motor domain with its lever bent strongly backwards. Lead heads attached at the 13 subunit spacing are 98 % in pre-powerstroke state, tethered there by the trail head. However, heads spaced by 11 subunits are more evenly distributed (60:40) pre- to post-powerstroke. No post-powerstroke lead heads are seen in heads spaced by 15 actin subunits. These results are consistent with an energy difference of 10 kJ/mole between the pre- and post-power stroke conformations at 11 and 13 actin subunit separation. The post-powerstoke lead head is a new attached state of myosin: the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. The rate of ADP dissociation from lead heads measured by stopped-flow fluorescence is >30 fold slower than from trail heads. The slower rate can be explained by a mechanism in which ADP only dissociates from the post-powerstroke state. ADP dissociation from the lead head is therefore inhibited by an unfavorable equilibrium between the pre-and post-powerstroke conformations. Supported by NIH EB00209.