A Subdomain Interaction at the Base of the Lever Allosterically Tunes the Mechanochemical Mechanism of Myosin 5a

Nikolett T Nagy,James R Sellers,Gábor M Harami,Mihály Kovács,Saikat Chakraborty,Takeshi Sakamoto

doi:10.1371/journal.pone.0062640

Abstract

The motor domain of myosin is the core element performing mechanochemical energy transduction. This domain contains the actin and ATP binding sites and the base of the force-transducing lever. Coordinated subdomain movements within the motor are essential in linking the ATPase chemical cycle to translocation along actin filaments. A dynamic subdomain interface located at the base of the lever was previously shown to exert an allosteric influence on ATP hydrolysis in the non-processive myosin 2 motor. By solution kinetic, spectroscopic and ensemble and single-molecule motility experiments, we determined the role of a class-specific adaptation of this interface in the mechanochemical mechanism of myosin 5a, a processive intracellular transporter. We found that the introduction of a myosin 2-specific repulsive interaction into myosin 5a via the I67K mutation perturbs the strong-binding interaction of myosin 5a with actin, influences the mechanism of ATP binding and facilitates ATP hydrolysis. At the same time, the mutation abolishes the actin-induced activation of ADP release and, in turn, slows down processive motility, especially when myosin experiences mechanical drag exerted by the action of multiple motor molecules bound to the same actin filament. The results highlight that subtle structural adaptations of the common structural scaffold of the myosin motor enable specific allosteric tuning of motor activity shaped by widely differing physiological demands.

Highlights

Myosins are ubiquitous molecular motors of eukaryotic cells that utilize the free energy from ATP hydrolysis for unidirectional translocation along actin filaments [1,2,3]
Earlier we found that the removal or inversion of the positive charges of the NTS lysine and the converter arginine (via the K84M and R704E mutations, respectively, in Dictyostelium discoideum (Dd) myosin 2) affected the lever priming step and, in turn, ATP hydrolysis and Pi release [15]
In line with earlier findings [9,17], wt-m5aS1 showed a significant increase in steady-state tryptophan (Trp) fluorescence emission upon interacting with ATP or the non-hydrolyzable ATP analog adenylyl-imidodiphosphate (AMPPNP) (Table 1). This signal change was earlier assigned to the ATP-sensitive tryptophan located in the so-called relay loop (W483 in m5a), which reports the postrigor-prepowerstroke transition coupled to ATP hydrolysis [8,18,19,20]

Summary

Introduction

Myosins are ubiquitous molecular motors of eukaryotic cells that utilize the free energy from ATP hydrolysis for unidirectional translocation along actin filaments [1,2,3]. The motor domain (MD) of myosin, the core element driving mechanochemical action, contains the actin and ATP binding sites as well as the base of the myosin lever (Fig. 1A) [4] The latter element amplifies structural changes occurring within the MD during the ATPase cycle to produce movement along actin. The binding of ATP to the actin-bound MD (step K1’ in Fig. 1B) weakens the actin affinity of different myosins by 3–6 orders of magnitude via allosteric changes (K2’) [5,6,7,8,9,10]. This process triggers the kinetic activation of the release of hydrolysis products Pi and ADP (K4’ and K5’, respectively), linked to the strengthening of the actomyosin interaction, and the swing of the lever leading to the powerstroke [13,14]

Methods

Results

Conclusion