Influence of lever structure on myosin 5a walking

Abstract

Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss.

Fig. 1.

(A) Wild-type myosin 5a walking in ATP. Also attached are two single NEM-treated myosin 2 heads, one of which is indicated (arrow). These demonstrate the polarity of the actin filament. (Scale bar, 50 nm.) (B–E) Montages of wild-type and mutant myosin 5a HMM molecules bound to actin in 0.5 μM ATP, all walking to the right. (B) 8IQ, (C) wild type, (D) 2Ala-6IQ, (E) 4IQ. (Scale bar, 25 nm.)

Fig. 2.

Details of stepping. Histograms show motor domain separation in raw images (gray bars) and image averages (black bars). Raw image distances were measured between motors using SPIDER. Distances between heads in image averages were measured between the attached actins. (Insets) Class averages obtained by segregating images according to motor separation. Classification used reference-free methods after windowing and aligning myosins attached by both heads. (Scale bar, 50 nm.)

Fig. 3.

Subclasses of (A) wild-type, (B) 6IQ+2Ala, and (C) 8IQ molecules bound 11, 13, and 15 actin subunits apart. (Upper) Straight lead levers in prepowerstroke conformation. (Lower) Curved lead levers, some attached to a motor in postpowerstroke conformation (asterisks). (Scale bar, 50 nm.)

Fig. 4.

Principal intermediates of the hydrolysis mechanism. Solid arrows joining states 1–5 indicate the principal processive pathway. Intermediates in the box show the prepowerstroke (1 and 2) to postpowerstroke (9 and 10) change. Dashed arrows (1→9→11→5) are a “futile pathway” that hydrolyzes ATP without stepping. Dotted arrows indicate side paths (5→8, 2→8, and 11→8) leading to dissociation and termination of processivity. Rapidly binding and dissociating lead heads are indicated by . T, ATP; D, ADP; P, phosphate.