Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin

Figures

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  Figure 1.. Structural model of K104 residue in M2β-S1. The location of the K104 residue in the RLC is depicted in the structural model of human M2β-S1 (HBCprestrokeS1 Homology Model, Spudich Lab, Stanford University, Stanford, CA). (A) M2β-S1 in the prestroke state with MHC (cyan), ELC (purple), and RLC (white) bound to the lever arm domain at respective IQ motifs (yellow). (B) Magnified view of the inset of RLC in A. Critical regions of the RLC molecule are highlighted, including phosphorylatable S15 (pink), the divalent cation binding loop (green, residues 37–48), and C terminus D166 (orange). The K104 residue is predicted to point away from the cyan lever arm in this model.

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  Figure 2.. Generation of myosin constructs. Illustration demonstrating our methodology to generate the various myosin constructs in the study. (A) Human M2β-S1 myosin (cyan) was generated via our C2C12 adenovirus recombinant system. C2C12 myoblasts are murine skeletal muscle cells, and thus the myosin contains the endogenous murine light chains (mELC and mRLC, pink and green, respectively). We performed an RLC exchange in order to study the impact of the human RLC (WT versus K104E, blue and red, respectively) on the mechanochemical properties of M2β-S1. Notably, the constructs still contain the endogenous mELC. (B) Full-length α-cardiac myosin (purple) was extracted from the ventricles of Tg animals expressing either human WT RLC (blue, n = 3) or human K104E RLC (red, n = 3). The image of the virus, petri dish, and mouse are under public domain (CC0). The image of the heart (titled “Heart oblique external”) was illustrated by Patrick J. Lynch; the original image was rotated slightly, and the atria and great vessels were made transparent. The original work and license (CCBY2.5) for the heart image can be found at the following link:…

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  (Scheme 1).

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  Figure S1.. SDS-PAGE of RLC exchange. Representative SDS-PAGE gel of protein preparation run in precast NuPage Novex 4–12% Bis-Tris, 1.0 mm, 10- or 12-well gels, demonstrating quality of M2β-S1 purification and RLC exchange. Lanes were loaded with the following samples: (1) mol wt marker (BioRad Precision Plus Protein Standard: 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kD, respectively), (2) stripped endogenous mRLC, (3) M2β-S1 WT human RLC (hRLC), (4) M2β-S1 K104E hRLC, (5) His-tagged hRLC control, and (6) M2β-S1 nonexchanged control. See labels for S1, endogenous murine RLC (mRLC) and ELC (mELC), as well as the exchanged hRLC. Note differential migration between His-tagged hRLC (19,589 D) and mRLC (18,864 D).

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  Figure S2.. Quantitative Western blot for hRLC exchange efficiency. Representative Western blot used to quantitate human RLC (hRLC) exchange in M2β-S1. Image is a representative Western blot (primary antibody: α-HIS; secondary antintintibody: α-mouse 680 nm) used to calculate the efficiency of the RLC exchange. WT and K104E M2β-S1 were loaded in decreasing amounts and compared with a RLC standard curve. Lanes of the SDS-PAGE gel were loaded with the following samples: (1) mol wt marker (BioRad Precision Plus Protein Standard: 250, 150, 100, 75, 37, 25, 20, 15, and 10 kD, respectively), (2–6) decreasing amounts of His-tagged hRLC standard protein (564, 423, 282, 141, and 70.5 ng WT RLC, respectively), (7–9) decreasing amounts of M2β-S1 WT RLC (20, 10, and 5 µl, respectively), and (10–12) decreasing amounts of M2β-S1 K104E RLC (20, 10, and 5 µl, respectively). Concentration was calculated by gel densitometry and compared with Bradford assay of total M2β-S1 concentration as a measure of exchange efficiency. Blots from three protein preparations were averaged to calculate heavy chain:light chain ratio (± SD): 0.97 ± 0.11 M2β-S1:WT RLC and 0.96 ± 0.11 for M2β-S1 K104E RLC.

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  Figure 3.. Actin-activated ATPase. The actin-activated ATPase activity of M2β-S1 WT RLC versus K104E RLC (0.1 µM) was examined as a function of actin concentration in MOPS 20 buffer at 25°C. The ATPase activity is plotted as a function of actin concentration and fit to a hyperbolic relationship to determine maximal ATPase and actin concentration at which ATPase is one-half maximal. The results represent an average of three experiments from three protein preparation (mean ± SD). See Table 1 for a summary of ATPase parameters.

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  Figure 4.. ATP-induced dissociation from actin. The experiment was performed by mixing a complex of M2β-S1 and pyrene actin with increasing concentrations of ATP (5–125 µM). (A and B) The fluorescence transients were best fit by a single exponential function at all ATP concentrations. Representative fluorescence transients at 5 µM ATP (A; kobs = 36.0 ± 0.2 and 31.1 ± 0.3 s–1 in WT and K104E, respectively) and 125 µM ATP (B; kobs = 553.6 ± 18.5 and 403.3 ± 12.0 s–1 in WT and K104E, respectively). (C) The second-order rate constant K’1Tk’+2 was determined from the linear-dependence on ATP concentration at low ATP concentrations (5–20 µM). (D) The maximal rate of ATP-induced dissociation (k’+2T) was determined from the hyperbolic fit of kobs as a function of ATP concentration. AU, arbitrary unit.

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  Figure 5.. ADP-release from actomyosin. Representative fluorescence transients of ADP release experiments using mantADP or pyrene actin. (A) We monitored mantADP dissociation by mixing acto–M2β-S1 complexed with mantADP with saturating ATP (1 mM), and the fluorescence transients were fit to a single exponential function (WT, kobs = 228.3 ± 3.8 s–1, and K104E, kobs = 297.5 s–1). (B) We examined ADP dissociation by mixing pyrene acto–M2β-S1.ADP with saturating ATP (2 mM), and the fluorescence transients were fit to a single exponential function (WT, kobs = 215.8 ± 8.9 s–1, and K104E, kobs = 145.0 ± 2.5 s–1).

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  Figure 6.. SuperPlots of in vitro motility assay from β-cardiac and α-cardiac myosin. (A) SuperPlot demonstrating 100 randomly sampled actin filament velocities analyzed by FAST (transparent circles, 100 of >2,500 filaments/experiment) and means from three experiments from distinct protein preparations (triangles) at 0.5 µM β-cardiac myosin. Student’s t test of sample means (n = 3) showed no difference (P = 0.8875) between WT (1,035 ± 53) and K104E (1,043 ± 20) in M2β-S1 constructs. (B) SuperPlot demonstrating 90 actin filament velocities analyzed manually using MTrackJ (transparent circles, 30 filaments/experiment) and means from experiments from three distinct ventricular myosin extractions (triangles) per group of Tg animals at 0.5 µM α-cardiac myosin. Student’s t test of sample means (n = 3) demonstrated a significant difference (*, P = 0.0176) between Tg WT (2,939 ± 278) and Tg K104E (4,194 ± 163) animals.

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  Figure S3.. Density dependence of in vitro motility of human β-cardiac myosin. Actin sliding velocities from the in vitro motility assay depicted at increasing densities of biotinylated M2β-S1 adhered to a streptavidin-nitrocellulose–coated glass coverslip. For each density, filament velocities analyzed by FAST tracking software from 3 or 4 videos were compiled between two protein preparations. Videos with >20% stuck filaments were excluded. Error bars represent mean ± SD. Actin sliding velocities are similar between M2β-S1 WT and K104E across all myosin densities, with <13% difference at each density.

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  Figure S4.. Structural model of K104E mutation in M2α-S1. The effect of the K104E mutation in the RLC is depicted in the structural model of modified M2α-S1 (HBCprestrokeS1 Homology Model, Spudich Lab, Stanford University, Stanford, CA). (A) The murine α-MHC (Myh6, cyan) and the murine RLC (Myl1, purple) were threaded through the model of human cardiac S1 (http://spudlab.stanford.edu/homology-models) using Chimera Modeller (zDOPE scores = −0.47 and −0.71, respectively). The human isoform of K104E (red) was threaded through and overlaid upon the human WT isoform (blue). The IQ motifs (yellow) represent the sites of light chain binding to the lever arm. (B) Magnified view of overlaid WT and K104E RLC from inset in A. The N-terminal lobe of K104E is not predicted to shift in the model (left, nearly perfect overlap), while the C-terminal lobe shifts significantly (right), such that K104E is more closely associated to the lever arm. Yellow dotted lines indicate distance from functional groups of lysine (K104, blue) and glutamate (K104E, red) to nearby carbon of isoleucine, the first residue of the IQ motif, which measure 11.0 Å and 4.5 Å, respectively.