Similarities and differences between frozen-hydrated, rigor acto-S1 complexes of insect flight and chicken skeletal muscles

Abstract

The structure and function of myosin crossbridges in asynchronous insect flight muscle (IFM) have been elucidated in situ using multiple approaches. These include generating "atomic" models of myosin in multiple contractile states by rebuilding the crystal structure of chicken subfragment 1 (S1) to fit IFM crossbridges in lower-resolution electron microscopy tomograms and by "mapping" the functional effects of genetically substituted, isoform-specific domains, including the converter domain, in chimeric IFM myosin to sequences in the crystal structure of chicken S1. We prepared helical reconstructions (approximately 25 A resolution) to compare the structural characteristics of nucleotide-free myosin0 S1 bound to actin (acto-S1) isolated from chicken skeletal muscle (CSk) and the flight muscles of Lethocerus (Leth) wild-type Drosophila (wt Dros) and a Drosophila chimera (IFI-EC) wherein the converter domain of the indirect flight muscle myosin isoform has been replaced by the embryonic skeletal myosin converter domain. Superimposition of the maps of the frozen-hydrated acto-S1 complexes shows that differences between CSk and IFM S1 are limited to the azimuthal curvature of the lever arm: the regulatory light-chain (RLC) region of chicken skeletal S1 bends clockwise (as seen from the pointed end of actin) while those of IFM S1 project in a straight radial direction. All the IFM S1s are essentially identical other than some variation in the azimuthal spread of density in the RLC region. This spread is most pronounced in the IFI-EC S1, consistent with proposals that the embryonic converter domain increases the compliance of the IFM lever arm affecting the function of the myosin motor. These are the first unconstrained models of IFM S1 bound to actin and the first direct comparison of the vertebrate and invertebrate skeletal myosin II classes, the latter for which, data on the structure of discrete acto-S1 complexes, are not readily available.

Figure 1

Surface views of one 360° helical repeat (~720Å) of rigor acto-CSk S1 (A) acto-wt Dros S1 (B), acto-IFI-EC S1 (C), and acto-Leth (D) density maps. The acto-S1 complexes share similar features including identical actin-motor domain positions and a long (~85 Å) LCD projecting away from the actin filament, labeled in panel A: MD - motor domain, ELC- essential light chain region, RLC-regulatory light chain region. Protein Purification. Skeletal myosin was prepared from chicken pectoralis muscle as previously described . Myosin from insect IFM was purified from either half of a single Leth thorax or ~ 300 wt Dros or 450–500 IFI-EC thoraces (per myosin preparation) as previously described . The wt Dros myosin yield was ~ 3.5 µg/fly where as the routine myosin yield from one Leth half thorax was ~ 500 µg. F-actin was prepared from rabbit muscle as previously described . S1 Preparation. The papain-Mg²⁺ myosin chicken S1 fragment was prepared as previously described with the following modifications. Briefly, myosin at 5 – 20 mg/ml was suspended in 0.2 M ammonium acetate (pH 7.0), 2 mM MgCl₂, and 1 mM DTT. The protein was digested with 0.03 mg papain (Worthington Biochemicals, Lakewood, NJ-activated at 1 mg/ml as per manufacturers instructions) per ml of myosin solution for 8 min. at 25°C. The reaction was stopped with 3 mM iodoacetic acid with one mini protease inhibitor tablet added/10ml solution (Roche Biochem, Mannheim, Germany) and placed on ice. The suspension was subjected to ultra-centrifugation (218 K × g) for 30 min. at 4°C to pellet the undigested myosin and rod while the soluble S1 fragment remained in solution. Insect S1 was prepared in a similar manner with the following exceptions: whole myosin was resuspended at 2 – 4 mg/ml in 2 mM KCl, 3 mM EDTA, pH 7.0, 20 mM potassium phosphate, pH 6.7, 5 mM β-mercaptoethanol and 5 mM MgCl₂ and digested with papain at 1:1000 w/w, enzyme to total protein, activated as described above, for 14–17 min. at 25°C. Longer digestion times yielded more S1 but resulted in significant digestion of the regulatory light chain. Concentration was determined using the E^1%₂₈₀ = 7.5, molecular weight of ~90 KD . S1 purity was assessed by SDS-PAGE. Acto-S1 complex preparation. Rabbit F-actin (0.05 mg/ml) was decorated with rigor (nucleotide free) S1 on holey copper carbon-coated grids (400 mesh, Ted Pella, Inc., Redding, CA, or 400 mesh Quantifoil, Structure Probe, Inc., West Chester, PA). Specimens were frozen in liquid ethane and stored in liquid nitrogen. Cryo-electron microscopy and image analysis. Specimens were examined with a Philips CM200 FEG (120 kV, 36K × magnification, −178°C). Images were acquired at 1.5–1.8 .m under focus using low dose conditions on Kodak SO-163 film. Fully decorated filaments were scanned (Perkin Elmer 1010 scanner) and subjected to helical image analysis using Phoelix . See Table 1 for a summary of the data and image processing results. S1 modeling was done manually using the program O and the figures were created with AVS (Advanced Visual Systems, Inc., Waltham, MA).

Figure 2

(A) Stereo, longitudinal view of the wt Dros (red) and Leth (cyan) insect acto-S1 maps superimposed in wire frame. Stereo images were prepared used MacPyMol (DeLano Scientific). Density is identical in the insect maps through the motor domain and the ELC region of the LCD. (B) Comparison of the wt Dros (red) and IFI-EC chimera (pink) maps. When viewed longitudinally, in stereo, a prominent spread of density in the RLC region of the IFI-EC chimera map is apparent. This very noisy “whale tail” suggests the RLC region of chimeric S1 adopts multiple positions. (C). Longitudinal view of the all of the insect maps. The similarity of the insect maps is emphasized by truncating the maps after the ELC omitting the “noisy” RLC region. wt Dros, red, Leth, cyan, IFI-EC, white.

Figure 3

Stereo, longitudinal view of acto-CSk S1 map (blue surface) superimposed with (A) wt Dros wireframe (red) and (B) Leth wireframe (cyan) maps shows the non-overlap regions. Stereo images were prepared used MacPyMol (DeLano Scientific). (C) CSk (blue wireframe) superimposed with wt Dros (red), IFI-EC (white) and Leth (cyan) maps truncated after the ELC.

Figure 4

(A) Nucleotide-free, chicken skeletal S1 crystal structure (yellow) docked on actin (green) modeled into the acto-CSk S1 map (blue, wireframe) in stereo, longitudinal view. Stereo image was prepared using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco. The RLC region of the crystal structure protrudes from the S1 envelope. (B) Cross-section and (C) longitudinal views of all of the density maps in wire frame superimposed, CSk (blue), wt Dros (red), Leth (cyan), IFI-EC (white), and compared to the nucleotide-free, chicken crystal structure (yellow) docked on actin (green) in the actoS1 reconstructions. Truncation of the insect maps after the ELC emphasizes that the ELC of the crystal structure is veering off the straight path of the IFM envelope toward the bend of the chicken RLC.