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

J Mol Biol. 2020 Jan 17;432(2):427-447. doi: 10.1016/j.jmb.2019.11.013. Epub 2019 Nov 29.

Abstract

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.

Keywords: Molecular dynamics; Motor proteins; Protein engineering; Skeletal muscle; X-ray crystallography.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence / genetics
  • Animals
  • Crystallography, X-Ray
  • Drosophila melanogaster / chemistry
  • Drosophila melanogaster / ultrastructure
  • Molecular Dynamics Simulation
  • 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 Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / ultrastructure*
  • Protein Structure, Tertiary
  • Skeletal Muscle Myosins / chemistry
  • Skeletal Muscle Myosins / genetics
  • Skeletal Muscle Myosins / ultrastructure*

Substances

  • Myosin Light Chains
  • Protein Isoforms
  • Skeletal Muscle Myosins
  • Myosin Heavy Chains