Alternative Exon 9-Encoded Relay Domains Affect More than One Communication Pathway in the Drosophila Myosin Head

Abstract

Summary We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced acto-myosin dissociation rate constant (K1k+2) and ADP affinity (KAD), measured for the chimeric constructs IFI-9b and EMB-9a compared to wild-type IFI and EMB. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50 kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of acto-myosin cross-bridge kinetics.

Alternative Relay and Converter Domains Tune Native Muscle Myosin Isoform Function in Drosophila

“…19 Modeling by Fisher et al 14,25-29 suggests that repositioning of the lever arm feeds back through the adjacent converter to the relay domain. Bloemink et al 18 used molecular modeling to demonstrate that specific residues could interact between the converter and relay. Interactions between alternative residues in the relay and converter domains in Drosophila myosin isoforms could be responsible for some differences in muscle physiology.…”

“…1A and B), which is hypothesized to communicate among the ATP-binding pocket, the actin-binding site and the converter domain. 13-18 The five alternative versions of exon 11 encode the central portion of the converter domain (Fig. 1A and C).…”

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function

“…The R759E mutation also disrupts flight ability, reduces myofibril stability, and diminishes muscle fiber power generation (20,21). Our molecular modeling studies identified specific residues in the relay domain (Asn 509 and Asp 511 ) that are likely to interact with converter domain residue Arg 759 during the pre-and postpower stroke states (22). Furthermore, relay residue Ile 508 can be crosslinked to converter residue Arg 759 in Dictyostelium discoideum myosin without severe functional defects (23), suggesting that it can serve as a linker to the converter domain.…”

“…1A, blue). Molecular modeling indicated that residues 508 -511 in the relay loop are located near the converter residue 759 (20) and defined weak and strong interactions of residues 509 and 511 during the rearrangements of the relay loop that are affiliated with the mechanochemical cycle (22). Furthermore, Ile 508 can be cross-linked to Arg 759 in Dictyostelium non-muscle myosin II when they are each substituted by cysteine (23).…”

“…However, residues 509 and 511 vary among the three exon 9-encoded regions (41). This suggests that alternative versions of these residues may modulate relay-converter interaction (22).…”