Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

Marieke J Bloemink,Karen H Hsu,Michael A Geeves,Sanford I Bernstein

doi:10.1074/jbc.ra120.014684

Abstract

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Highlights

Muscle myosin isoforms display a large variety in kinetic properties and force production, despite their sequences being highly conserved
The EMB myosin isoform is found in the embryonic body wall muscle, which is used for slow locomotion of the larvae, whereas the indirect flight muscle myosin isoforms (IFI) myosin is present in the muscle that can generate very high wing beat frequencies and enables flight
Transgenic expression of EMB in the indirect flight muscle resulted in loss of flight ability [3], and subsequent studies using isolated muscle fibers and/or myosin proteins confirmed the striking differences in kinetic and mechanical properties between IFI and EMB myosin isoforms [4,5,6]

Summary

Introduction

Muscle myosin isoforms display a large variety in kinetic properties and force production, despite their sequences being highly conserved. Previous work found that exchange of the exon 3 regions between IFI and EMB significantly changes the steady-state kinetic properties of both Drosophila muscle myosin isoforms.

Results

Conclusion