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

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Abstract

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 actomyosin 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 values. 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 actomyosin cross-bridge kinetics.

Introduction

The myosin superfamily consists of at least 24 classes of ATP-dependent motor proteins that interact with actin filaments and are involved in a large number of physiological processes, such as muscle contraction, phagocytosis, cell motility, and vesicle transport.1, 2 All myosins appear to undergo the same ATP-driven cycle of interaction of myosin with actin, known as the cross-bridge cycle, yet myosins show a wide variety of different mechanical activities. Class II myosins consist of two heavy chains (MHCs) and two pairs of light chains: the regulatory light chains (RLCs) and essential light chains (ELCs). The C-termini of the myosin heavy chains dimerize as a coiled coil (“myosin tail”), whereas the N-termini form the two myosin “heads” or “motor domains.” The most familiar activity of class II myosins is muscle contraction.

The various isoforms of Drosophila melanogaster muscle MHC are encoded by a single gene (Mhc).3 Alternative RNA splicing produces myosin isoforms with different tissue specificity and functional properties. Of the 19 exons in Mhc, 5 exon sets are alternatively spliced, while exon 18 is either included or excluded. Four of the six alternative exon sets encode portions of the motor domain.4 The domains encoded by exon 3 (amino acid residues 69–116) and exon 9 (472–528) are located near the sulfhydryl helix, a part of the myosin head that is thought to transduce the chemical energy provided by ATP hydrolysis into movement. The exon 7 domain (301–335) is near the ATP-binding pocket. The exon 11 region (724–764) is part of the converter domain, providing a bridge between the long relay helix encoded by exon 9 and the ELC. Exon 11 encodes the lowest degree of conservation among the alternative regions (38%), followed by exon 7 (56%), exon 3 (75%), and exon 9 (89%).

Exon 9 encodes one of the variable regions in the myosin head, whose location is indicated in Fig. 1a. This region is also known as the “relay helix–loop–helix domain” or “relay domain” and plays a crucial role in the structural coupling between ATP hydrolysis and the recovery stroke in the myosin motor. The mechanism of this coupling has recently been modeled in detail based on two crystal structures of Dictyostelium myosin II representing the prepower stroke state and the postpower stroke state.5 It was proposed that before ATP hydrolysis can occur, the switch-2 loop closes in a stepwise fashion. Closure of switch-2 is linked to an initial rotation of the converter domain via a seesaw pivoting of the relay helix (step 1), whereas the second phase of converter rotation is coupled to the final closing of switch-2 via the SH1 and SH2 helices and the wedge loop (step 2). This proposed coupling mechanism is consistent with the many available myosin crystal structures.6, 7

The Drosophila relay domain is encoded by alternative exons 9a, 9b, or 9c.3 Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, while exon 9b encodes a relay domain found within one of the embryonic body wall (EMB) isoforms.8 The amino acid sequences of the two relay domains, encoded by exon 9a (IFI) or exon 9b (EMB), are depicted in Fig. 1b and correspond to residues 472–528 of chicken myosin II (or residues 469–525, using Drosophila numbering). For reference purposes, we use the chicken numbering throughout this article. We previously generated chimeric myosins (IFI-9b and EMB-9a) by exchanging relay domains between the two native isoforms.9 This allows for characterization of functional differences ascribed to alternative versions of this domain. Interchanging the two versions of exon 9 resulted in different effects on the indirect flight muscle (IFM) ultrastructure and performance. IFI-9b flies displayed wild-type structure and stability of IFM myofibrils, while EMB-9a flies showed a significant disruption of muscle structure and myofibril stability when compared to EMB flies. Flight and jump ability tests of flies expressing the IFI-9b myosin isoform were near wild type, while flies expressing the EMB-9a isoform failed to rescue the flightless phenotype and impaired jumping phenotypes observed in flies expressing the EMB isoform. Results from biochemical and mechanical experiments performed on full-length IFI-9b myosin molecules showed that its basal and actin-stimulated ATPase activities, as well as its in vitro actin sliding velocity, are similar to those of IFI.9 In contrast, the EMB-9a myosin isoform shows a reduction of both basal and actin-stimulated ATPase activities compared to EMB, a marked increase in actin affinity, and lacks the ability to translocate actin filaments in vitro.9

This article describes steady-state and transient kinetics measurements using S1 fragments of the previously generated exon 9 (IFI-9b and EMB-9a) chimeric MHC isoforms to investigate the roles of the alternative relay domains in regulating the actomyosin cross-bridge cycle. Our results show that several kinetic parameters are affected, including ADP affinity, actin affinity, and ATP-induced actomyosin dissociation. These results indicate that the relay domain, encoded by exon 9, affects more than one signal transduction pathway in the Drosophila myosin head. In addition to the expected effects on the communication pathway between the nucleotide-binding pocket and the converter domain, the signal transfer from the nucleotide-binding pocket toward the actin-binding site is altered when relay domains between IFI and EMB are exchanged. Homology models of the chimeric myosin S1 molecules suggest a possible mechanism by which exchanging the relay domain can alter the kinetic properties of the myosin head.

Section snippets

Actin-activated Mg2+-ATPase of the S1 fragments of IFI, EMB, IFI-9b, and EMB-9a

A summary of the steady-state properties of the S1 fragments of wild-type and chimeric myosins is shown in Table 1. This represents the first determination of these kinetic parameters for Drosophila myosin S1 ATPase. In agreement with our previous data for full-length wild-type myosins,9, 10 basal Mg2+-ATPase and actin-activated ATPase values (Vmax) were significantly higher for IFI S1 (0.074 ± 0.016 and 2.47 ± 0.29 s 1, respectively) compared to EMB S1 (0.016 ± 0.002 and 0.67 ± 0.06 s 1,

Discussion

In this study, we utilized S1 fragments of wild-type (IFI and EMB) and chimeric (IFI-9b and EMB-9a) myosins to continue our analysis of the contribution of alternative exons encoding the Drosophila myosin head to myosin isoform functional differences. This study focused on the structure and function of alternative relay domains encoded by exon 9. We determined the steady-state kinetic parameters of the actin-activated ATPase activities and examined the transient kinetics of the cross-bridge

Generation of subfragment-1 (S1) from isolated full-length myosin

Myosin was isolated from the IFM of 170–250 wild-type or transgenic flies (those expressing the IFI, EMB, EMB-9a, or IFI-9b myosin isoforms in the IFM) as previously described.36 The production of S1 by α-chymotrypsin digestion was carried out using a method based on that of Silva et al.37 with the following modifications: The final myosin pellets obtained after centrifugation of a myosin solution in low-salt buffer were dissolved in 20 μl of high-salt digest buffer [480 mM NaCl, 20 mM Na2PO4,

Acknowledgements

This work was supported by NIH grant GM32443 (to S.I.B.) and Wellcome Trust grant 070021 (to M.A.G.). We thank Martin Webb (National Institute for Medical Research, Mill Hill, London, UK) for the coumarin ATP/ADP used in this work and Anju Melkani for excellent technical support.

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1

Present address: C. M. Dambacher, Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, SR207, La Jolla, CA 92037, USA.

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