N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size

Abstract

Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ~19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method.

Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift

“…Similar results were obtained using the assay for adult zebrafish skeletal myosin step-size and step-frequency [11]. ELC N-terminus binding to actin is the mechanism for generating the 3 unitary steps [5] that is modeled in a 4-pathway network in Fig 8.…”

“…Comparing ensemble average and single myosin results we find there are three critical distinctions. We report that the average step-size shortens under increasing load due to shifting frequencies among short, intermediate, and long unitary step-sizes, we directly observe the force bearing 0 length step, and the mechanism for how all of it is accomplished involving ELC N-terminus actin binding follows directly from earlier in vitro single myosin measurements [5, 17]. The latter indicates that shifting step-frequencies to favor the short step is a downshifting maneuver caused by the ratcheting effect of the actin bound ELC N-terminus also showing that the in vitro and in vivo systems closely correlate.…”

In vitro actin motility velocity varies linearly with the number of myosin impellers

“…No change in measured step-size implies compliance in the myosin linkage is negligible while compliance due to intrinsic myosin elasticity is undetectable because the α-actinin generally kinetically detaches from actin during the course of the myosin power stroke. We found no significant change in the step-sizes due to the loading of βmys implying negligible assay compliance [2]. Experimental conditions in this previous work were identical to those used here.…”

“…We previously evaluated assay compliance by assessing βmys step-size under load imposed by α-actinin [2]. There the fraction of moving actin filaments vs α-actinin concentration in a βmys motility assay indicated a linear decline for increasing α-actinin except at the highest concentrations where it was unable to fully inhibit actin filament sliding.…”

“…The strong actin binding head in any ADL model will resist filament sliding over distances larger the average unitary cardiac myosinstep-sizeof˜6nmsince any larger compliance would disable the myosin motor from moving an actin filament in the motility assay. In vitro motility assay compliance for the assay and motor used here is a known characteristic [2]. It further constrains the ADL model to resisting all measured sliding distances equal to or larger than 0.5 nm or ˜1/12 of a myosin unitary step-size.…”