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

Q: What are the key findings of this study?

Exchanging the exon 3 region between Drosophila IFI and EMB myosin isoforms completely reversed the ADP release rate compared to wild-type.

Q: What is the clinical evidence from this study?

Population: Drosophila melanogaster muscle myosin heavy chain II isoforms. Intervention: Exon 3 chimeric myosin S1 constructs (IFI-3a and EMB-3b) vs. Wild-type Drosophila myosin S1 isoforms (IFI and EMB). Primary outcome: ADP release rate in the absence of actin (k-D) and ATP-induced dissociation of acto-S1 (K1k+2) (p=<0.007).

Q: What does this research mean for the field?

Exchanging the alternative exon 3-encoded N-terminal region between Drosophila embryonic and indirect flight muscle myosin isoforms reverses the ADP release rate and modulates crossbridge kinetics via a communication pathway between the purine binding loop and essential light chain. Novelty: ClaimNovelty.INCREMENTAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

Q: What question did this study set out to answer?

To examine the biochemical and biophysical effects of alternative exon 3-encoded regions on Drosophila myosin catalytic activity and inter-domain communication.

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

doi:10.1074/jbc.ra120.014684

August 19, 2020Journal of Biological ChemistryOpen Access

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

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Key result

Exchanging myosin exon 3 completely reverses isoform ADP release rates.

P<0.007

Why the study?

The study investigated the biochemical and biophysical properties of an alternative exon-encoded region within the Drosophila myosin catalytic domain.

Does exchanging the alternative exon 3-encoded N-terminal region between fast (IFI) and slow (EMB) Drosophila myosin isoforms alter actomyosin cross-bridge kinetics and communication between the nucleotide binding pocket and the essential light chain?

Key Points

To examine the biochemical and biophysical effects of alternative exon 3-encoded regions on Drosophila myosin catalytic activity and inter-domain communication.
Engineered chimeric myosin S1 constructs (IFI-3a and EMB-3b) by exchanging alternative exon 3 regions between slow embryonic (EMB) and fast indirect flight muscle (IFI) isoforms.
Measured basal and actin-activated ATPase activities, ADP release rates without actin (k-D), actin-bound ADP affinity (KAD), and ATP-induced acto-S1 dissociation (K1k+2).
Conducted homology modeling based on an established Drosophila EMB crystal structure to trace structural pathways linking the purine binding loop and essential light chain.
ADP release rates (k-D) in the absence of actin were completely reversed for both chimeric constructs relative to their native counterparts.
Basal and actin-activated ATPase activities shifted reciprocally, decreasing in IFI-3a compared with wild-type IFI and increasing in EMB-3b compared with wild-type EMB.
ATP-induced acto-S1 dissociation (K1k+2) was reduced in both chimeras, whereas actin-bound ADP affinity (KAD) was unaltered in IFI-3a and increased toward IFI levels in EMB-3b.

Structured PICO

Does exchanging the alternative exon 3-encoded N-terminal region between fast (IFI) and slow (EMB) Drosophila myosin isoforms alter actomyosin cross-bridge kinetics and communication between the nucleotide binding pocket and the essential light chain?

P

Population

In vitro biochemical, kinetic, and homology modeling study of purified Drosophila melanogaster slow embryonic (EMB) and fast indirect flight muscle (IFI) myosin S1 isoforms and exon 3 chimeras.

I

Intervention

Chimeric myosin S1 constructs (IFI-3a and EMB-3b) generated by exchanging the alternative exon 3a and 3b encoded regions (residues 69-116, part of the N-terminal beta-barrel) between native EMB and IFI myosin isoforms.

C

Comparator

Native wild-type slow embryonic body wall (EMB, containing exon 3a) and fast indirect flight muscle (IFI, containing exon 3b) myosin S1 isoforms.

O

Outcome

Biochemical and biophysical kinetic properties of the myosin S1 head, including ADP release rate in the absence of actin (k-D), ADP affinity in the presence of actin (KAD), ATP-induced dissociation of acto-S1 (K1k+2), and basal and actin-activated ATPase activity.surrogate

Main Result

Absolute Event Rate: 2.1% vs 7%

p-value: p=<0.007

The variable exon 3-encoded N-terminal domain of myosin forms a critical communication relay between the purine-binding loop and essential light chain via helix NT, regulating cross-bridge kinetics and explaining how human beta-cardiac myosin mutations along this pathway may cause cardiomyopathy.

Limitations

The EMB crystal structure used as a template is only available for a rigor-like configuration with no nucleotide bound, requiring homology modeling for other states.
Reversal of the fluorescence change upon cmADP release in EMB-3b currently has no explanation and requires analysis of a high-resolution structure to resolve.
The Drosophila EMB crystal structure is only available for a rigor-like configuration without bound nucleotide, necessitating homology models derived from scallop myosin structures for other cross-bridge states.
The cause of the reversed directionality of the fluorescence trace upon cmADP release in EMB-3b is unknown and requires a high-resolution structure to resolve.

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Implication

Exon 3 regulates myosin ADP release kinetics in Drosophila; leaves open relevance to human beta-cardiac myosin mutations in cardiomyopathy.

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 ( K AD ) 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 ( K 1 k +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.

Discussion

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Cite This Study

Bloemink et al. (2020) studied Drosophila melanogaster muscle myosin heavy chain II isoforms. Exon 3 chimeric myosin S1 constructs (IFI-3a and EMB-3b) vs. Wild-type Drosophila myosin S1 isoforms (IFI and EMB) was evaluated on ADP release rate in the absence of actin (k-D) and ATP-induced dissociation of acto-S1 (K1k+2) (p=<0.007). Exchanging the exon 3 region between Drosophila IFI and EMB myosin isoforms completely reversed the ADP release rate compared to wild-type.

synapsesocial.com/papers/6a8c17ad9ac394ad33caca7e https://doi.org/10.1074/jbc.ra120.014684

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

Key Points

Structured PICO

Main Result

Limitations

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Implication

Abstract

Discussion

Cite This Study

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

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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