X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties

Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaste...

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Bibliographic Details
Published in:Journal of molecular biology 2020-01, Vol.432 (2), p.427-447
Main Authors: Caldwell, James T., Mermelstein, Daniel J., Walker, Ross C., Bernstein, Sanford I., Huxford, Tom
Format: Article
Language:English
Subjects:
Amino Acid Sequence - genetics
Animals
Crystallography, X-Ray
Drosophila melanogaster - chemistry
Drosophila melanogaster - ultrastructure
Molecular dynamics
Molecular Dynamics Simulation
Motor proteins
Myofibrils - genetics
Myofibrils - ultrastructure
Myosin Heavy Chains - chemistry
Myosin Heavy Chains - genetics
Myosin Heavy Chains - ultrastructure
Myosin Light Chains - chemistry
Myosin Light Chains - genetics
Myosin Light Chains - ultrastructure
Protein Domains - genetics
Protein engineering
Protein Isoforms - chemistry
Protein Isoforms - genetics
Protein Isoforms - ultrastructure
Protein Structure, Tertiary
Skeletal muscle
Skeletal Muscle Myosins - chemistry
Skeletal Muscle Myosins - genetics
Skeletal Muscle Myosins - ultrastructure
X-ray crystallography
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Summary:Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using our system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, we prepared and crystallized stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain. We solved the x-ray crystal structure by molecular replacement and refined the resulting model against diffraction data to 2.2 Å resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins. [Display omitted] •A 2.2 Å x-ray crystal structure of the embryonic isoform of Drosophila myosin II.•This is the first x-ray crystal structure of an insect myosin motor domain.•The model closely resembles rigor-like myosins from other species.•61 amino acids differ between embryonic and indirect flight muscle myosins.•Molecular dynamics reveal that relay and converter differ most between isoforms.
ISSN:0022-2836
1089-8638
1089-8638
DOI:10.1016/j.jmb.2019.11.013