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. 2016 Jan 12;55(1):186-98.
doi: 10.1021/acs.biochem.5b00817. Epub 2015 Dec 29.

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

Yihua Wang, Katalin Ajtai, Katarzyna Kazmierczak  1 , Danuta Szczesna-Cordary  1 , Thomas P Burghardt
Affiliations

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

Yihua Wang et al. Biochemistry. .

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.

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Figures

Figure 1
Figure 1
Sequences of vELC isoforms: porcine (NP_001265702, 197 residues) and its 17 amino acid N-terminal truncation, Δ17; mouse (P09542, 204 residues); human (P08590, 195 residues) expressed in mice in this study (WT) and its 43 amino acid N-terminal truncation mutant, Δ43. Sequences of human skeletal muscle ELC isoforms: A1 (NP_524144.1, 194 residues) and A2 (P06741, 150 residues). ELC(A2) is the model for Δ43. Following I60 are 144 residues not shown.
Figure 2
Figure 2
The proposed 3 step-size mechanism for cardiac myosin moving actin in vitro and the myosin dimers participating in motility. Panel a is the full lever-arm swing step of 8 nm with actin binding of the vELC N-terminus. Panel b shows the 5 nm step followed by detachment from actin (towards right) or an unlikely event when lingering ADP causes a subsequent 3 nm step (towards left). The full 8 nm step facilitated by the vELC/actin link corresponds to a lever-arm rotation ~19° larger than that for a 5 nm step. Angle, ∠cde in panel a, indicates the acute angle specifying lever-arm orientation relative to the actin filament. Panel c shows the myosin dimers participating in motility and notation representing the native homodimers (NHD) containing intact vELC, the heterodimer (HED) with one modified and one native vELC, and the modified homodimer (MHD) with two modified vELC’s. Mouse vELC is distinguished from human vELC by the tip-down (mouse) or tip-up (human) ELC N-terminus.
Figure 3
Figure 3
The Qdot assay event-velocity histogram (left most column) and the unitary step-frequency expectations (middle and right columns) for porcine Δ0 (row a), Δ17βmy@30% with 30% of the vELC’s truncated at residue 17 (row b), and Δ17βmy@60% with 60% of the vELC’s truncated at residue 17 (row c). Left column rows a-c: The event-velocity histogram for unitary step-size data (■ and dashed line) and simulation (solid line) at the low velocity end. Step-sizes correspond to the short (↑red), intermediate (↓green), and long, (↑blue) steps with associated numeric values in nm. Middle column rows a-c: Step-frequency expectation corresponds to the short (red), intermediate (green), and long (blue) step-sizes with numeric mean values ωS, ωI, and ωL ± standard deviation. Step-frequency expectations are derived from simulation of event-velocity histogram data as described in METHODS. Relative fractions of NHD, HED, and MHD are indicated next to their icon. Right column row c: Step-frequency expectations for pure species MHD (truncated vELC homodimer, solid lines) and HED (1 truncated and 1 native vELC heterodimer, dashed lines) are derived as explained in METHODS. Numeric mean values ωS, ωI, and ωL ± standard deviation represent the pure MHD species only. An independent experimental event-velocity histogram was obtained from each of 10–12 acquisitions × 3 separate protein preparations or 30–36 acquisitions for each isoform (Δ0, Δ17@30%, and Δ17@60%). The 30–36 best fitting independent simulations of these data were the basis for the standard deviation estimates indicated in the figure.
Figure 4
Figure 4
The Qdot assay event-velocity histogram (left most column) and the unitary step-frequency expectations (middle and right columns) for mouse NTg (row a), WTαmys@30% with 30% of the native mouse vELC replaced with intact human vELC (row b), and Δ43αmys@60% with 60% of the native mouse vELC replaced with human vELC truncated at residue 43 (row c). Left column rows a-c: The event-velocity histogram for unitary step-size data (■ and dashed line) and simulation (solid line) at the low velocity end. Step-sizes correspond to the short (↑red), intermediate (↓green), and long, (↑blue) steps with associated numeric values in nm. Middle column rows a-c: Step-frequency expectations correspond to the short (red), intermediate (green), and long (blue) step-sizes with numeric mean values ωS, ωI, and ωL ± standard deviation. Step-frequency expectations are derived from simulation of event-velocity histogram data as described in METHODS. Relative fractions of NHD, HED, and MHD are indicated next to their icon. Right column row b: Step-frequency expectations for pure species MHD (human vELC homodimer) and HED (1 human and 1 native vELC heterodimer) assuming Model 2 where MHD and HED are identical. Right column row c: Step-frequency expectations for pure species MHD (Δ43 human vELC homodimer) and HED (1 Δ43 human and 1 native vELC heterodimer) assuming Model 2 where MHD and HED are identical. An independent experimental event-velocity histogram was obtained from each of 10–12 acquisitions × 1 protein preparation or 10–12 acquisitions for each isoform (NTg, WT@30%, and Δ43@60%). The 10–12 best fitting independent simulations of these data were the basis for the standard deviation estimates indicated in the figure.
Figure 5
Figure 5
The ensemble averaged duty-ratio ( × 102) expectation for pure native vELC homodimers (NHD, dashed line with squares) and truncated vELC homodimers (MHD, thick line) of βmys (panel a) and αmys (panel b).
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