Journal of Molecular Biology
Alternative S2 Hinge Regions of the Myosin Rod Differentially Affect Muscle Function, Myofibril Dimensions and Myosin Tail Length
Introduction
Myosin II, the molecular motor responsible for muscle contraction, is a hexameric protein consisting of two myosin heavy chains (MHCs), two essential light chains and two regulatory light chains (Figure 1(a)). Each MHC monomer has an actin-binding N-terminal globular head, an alpha-helical rod, and a non-helical C-terminal tailpiece. Two MHC rods intertwine to form an alpha-helical coiled-coil. Charged regions on the surface of the coiled-coils allow myosin rods to electrostatically interact and polymerize into the thick filament backbone.1 Thick filaments interdigitate with actin-containing thin filaments, and these serve as the key contractile elements of striated muscle sarcomeres. During the contractile cycle, the catalytic myosin head hydrolyzes ATP and subsequently forms crossbridges with actin. The subfragment 2 (S2) portion of the myosin rod tethers the head to the thick filament. Just before the release of inorganic phosphate, crossbridges swing causing thin filaments to slide past thick filaments, resulting in sarcomere contraction.2
Assembly of MHC dimers and thick filaments depends on the sequence periodicity of the myosin rod.1 The rod contains a heptapeptide repeat (a, b, c, d, e, f, g), with hydrophobic residues concentrated at positions a and d. Hydrophobic interactions form a “seam” between the two alpha-helices of the coiled-coil, which is stabilized by salt-bridges between charged residues at positions e and g. Positions b, c and f are charged mainly at the surface of the rod and are free to interact with surrounding molecules. The heptad repeats can be further grouped into 40 zones of non-identical 28 residue repeats. Each 28 residue repeat contains alternating regions of positive and negative charge, causing myosin rods to assemble in a staggered arrangement within the thick filament.1,3 These 40 zones are interrupted at four positions by an additional “skip” residue, which breaks the heptad repeat1,3 and may serve to control the stagger of neighboring parallel myosin molecules during thick filament assembly.4
Myosin molecules are characterized by the presence of two “hinges” that lie C-terminal to the globular head (Figure 1(a)). The S1/S2 hinge is located at the junction of subfragment 1 (S1) and S2 and may help to position myosin heads interacting with the thin filament. The S2/LMM hinge region of the MHC rod is located between the C terminus of short S2 and the N terminus of light meromyosin (LMM), spanning ∼152 amino acid residues. Two of the four skip residues described above flank the S2/LMM hinge domain,5 and may correspond to the location of bends in the rod observed in electron micrographs of negatively-stained myosin molecules.6 The S2/LMM hinge is proposed to be a flexible domain that allows myosin S1 to lift away from the thick filament and reach toward the thin filament.7., 8., 9. Proteolysis of thick filaments suggests that the C-terminal two-thirds of the MHC rod form the thick filament backbone and the N-terminal third is loosely attached.1,10,11 The S2/LMM hinge may serve as a flexible linker connecting these regions, but it is important to note that the distinct bends in the myosin rod flank the S2/LMM hinge, while the hinge itself is not a known site of rod bending.
The S2/LMM hinge region has a reduced propensity to form a coiled-coil. Lu and Wong predicted that the coiled-coil formed from the rabbit skeletal MHC S2/LMM hinge is relatively unstable, since it is rich in basic residues and deficient in hydrophobic residues.12 The significantly lower stability of the S2/LMM hinge coiled-coil compared to other portions of the rod leads to preferential melting (helix to random-coil transition) when temperature, pH or ionic strength is perturbed. The helix to random-coil transition is implicated in shortening of the rod during temperature or pH alteration, with the principal melting site being the S2/LMM hinge.13,14 Recent experimental evidence supports the flexibility of this hinge region.[15], 16., 17.
The S2/LMM hinge region may influence mechanochemical energy transduction. By undergoing a helix-coil transition in vivo, the resulting shortening of the rod could contribute to force generation.18 Harrington and others obtained evidence of helix-coil transition in muscle fibers upon activation or when crossbridges are released from the thick filament backbone, yielding a more open and proteolytically accessible state.19., 20., 21., 22., [23], 24. Antibodies to S2 or the hinge greatly reduce isometric force generation, decrease muscle stiffness, suppress the movement of actin filaments in in vitro motility assays and reduce active shortening of sarcomeres, without altering the Mg-ATPase activity of myofibrils.25,26
Differences in S2/LMM hinge sequences among myosin isoforms correlate with muscle-specific properties, implying that this domain may define some aspects of muscle function. Sequence differences in the hinge region are among the relatively few residues that vary between rat alpha and beta-cardiac MHCs, which have unique enzymatic and mechanical properties.27,28 Thirteen non-conserved differences are located in the putative hinge and its immediate N-terminal region (residues 1075–1352). In scallop, the central region of this MHC hinge is encoded by alternative exons; one version is used in fast striated muscle, while the other is used in the relatively slow catch smooth muscle.29
Drosophila melanogaster MHCs also have isovariant S2/LMM hinge regions that correlate with muscle-specific properties. In Drosophila, alternative RNA splicing of five sets of alternative exons as well as inclusion or exclusion of an alternative 3′ exon yield functionally-different MHC isoforms that are expressed in a developmental and tissue-specific manner.30., 31., [32], 33. The central 26 amino acid residues of the S2/LMM hinge are encoded by one set of these alternative exons, 15a and 15b. For simplicity, we use the terminology hinge A and hinge B to describe the encoded regions; however, only 26 residues of the entire S2/LMM hinge regions differ (Figure 1(a)). The use of the alternative hinge regions correlates to muscle contraction speed.34,35 MHC isoforms in fast muscles (the synchronous jump muscle and the asynchronous indirect flight muscles) use exclusively hinge A.34,35 Only hinge B is expressed in slow muscle types, i.e. embryonic and adult body wall muscle. Transcripts containing exon 15a or 15b are both found in intermediate muscle types such as those in the legs and proboscis.34,35 Alternative versions of these hinges and/or variations in other regions of the myosin molecule may define the observed differences in contractile and structural properties of various muscle types.
To address the structural and functional roles of alternative S2/LMM hinges in Drosophila, we substituted hinge B for hinge A in MHC isoforms expressed in two fast muscle types, the indirect flight muscle (IFM) and the tergal depressor of the trochanter (TDT) or jump muscle. We found hinge B increased the length of the MHC rod, increased IFM sarcomere lengths, weakened myofibril stability, and impaired the function of the IFM and TDT compared to hinge A. Our results demonstrate that the Drosophila muscle myosin S2/LMM hinge region is important for myofibril assembly and stability, and that the slow hinge cannot functionally substitute in fast muscle myosin. Thus, differences in the S2/LMM hinge region of myosin are critical for dictating muscle-specific properties.
Section snippets
Hinge sequence analysis
We compared D. melanogaster S2/LMM alternative hinge regions A and B to define structural variations that could affect myosin function differentially (Figure 1(b)). Nineteen of 26 amino acid residues differ between the alternative hinge domains, and nine of these have hydrophobicity or charge changes. The net charge of the hinge A-specific domain is +1, while that of hinge B is –1. Hinge A has charged residues in six of the eight e and g positions, whereas hinge B has four. Hinge A has
Discussion
The strict use of alternative MHC S2/LMM hinges in different muscle types of Drosophila melanogaster suggests that the variable central domains of these hinges impart specialized properties to the muscles in which they are expressed. Hinge A is well conserved in other insects, including various Drosophila species and other dipterans such as a mosquito, but has a lower degree of conservation with muscle myosin IIs of other organisms (Figure 1(c)). It is unusual in that it is positively charged
DNA and protein sequence analyses
Drosophila Mhc genes were aligned using Multi-LAGAN67 and the database developed by the Eisen laboratory at the University of California, Berkeley‡. Alignments were kindly provided by Dr Venky Iyer. These were analyzed using the Jalview Java Alignment Editor.68 D. melanogaster hinge A and B sequences submitted to a BLAST search§ against the translated protein database (tblastn) produced hits to D. hydei (Genbank
Acknowledgements
We are grateful to Mr Allen Church and Dr Michelle Mardahl-Dumesnil for their technical expertise, and to Mr Gregory Aselis for initial work on the project. We thank Dr Jim Vigoreaux (University of Vermont) and Dr Sunita Patel (Brigham and Women's Hospital) for unpublished data and Dr Venky Iyer (University of California, Berkeley) for Drosophila Mhc gene alignments. We appreciate the advice of Dr Roger Craig (University of Massachusetts Medical School) and Dr Steve Barlow regarding sample
References (79)
- et al.
Periodic features in the amino acid sequence of nematode myosin rod
J. Mol. Biol.
(1983) - et al.
Skip residues and charge interactions in myosin II coiled-coils: implications for molecular packing
J. Mol. Biol.
(2005) - et al.
Negative staining of myosin molecules
J. Mol. Biol.
(1985) Skip residues correlate with bends in the myosin tail
J. Mol. Biol.
(1990)- et al.
Stability and melting kinetics of structural domains in the myosin rod
J. Mol. Biol.
(1983) - et al.
Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility
J. Mol. Biol.
(1977) - et al.
Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation
J. Mol. Biol.
(1969) - et al.
The amino acid sequence and stability predictions of the hinge region in myosin subfragment 2
J. Biol. Chem.
(1985) - et al.
Electron microscope study of the effect of temperature on the length of the tail of the myosin molecule
J. Mol. Biol.
(1986) - et al.
High flexibility of the actomyosin crossbridge resides in skeletal muscle myosin subfragment-2 as demonstrated by a new single molecule assay
J. Struct. Biol.
(2005)
Mechanical properties of single myosin molecules probed with the photonic force microscope
Biophys. J.
Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin
Biophys. J.
Crossbridge release and alpha-helix-coil transition in myosin and rod minifilaments
J. Mol. Biol.
An enzyme-probe study of motile domains in the subfragment-2 region of myosin
J. Mol. Biol.
Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge
J. Mol. Biol.
Local melting in the subfragment-2 region of myosin in activated muscle and its correlation with contractile force
J. Mol. Biol.
Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences
J. Mol. Biol.
Spatially and temporally regulated expression of myosin heavy chain alternative exons during Drosophila embryogenesis
Mech. Dev.
Prediction and analysis of coiled coil structures
Methods Enzymol.
Differential localization of two myosins within nematode thick filaments
Cell
Tissue-specific expression of the alternately processed Drosophila myosin heavy-chain messenger RNAs
Dev. Biol.
An alternative domain near the nucleotide-binding site of Drosophila muscle myosin affects ATPase kinetics
J. Mol. Biol.
Passive stiffness in Drosophila indirect flight muscle reduced by disrupting paramyosin phosphorylation, but not by embryonic myosin S2 hinge substitution
Biophys. J.
Shape and flexibility of the myosin molecule
J. Mol. Biol.
Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain
J. Mol. Biol.
Defects in the Drosophila myosin rod permit sarcomere assembly but cause flight muscle degeneration
J. Mol. Biol.
Alternative N-terminal regions of Drosophila myosin heavy chain tune muscle kinetics for optimal power output
Biophys. J.
Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity
J. Biol. Chem.
Length of myosin rod and its proteolytic fragments determined by electron microscopy
FEBS Letters
Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle
Nature
Coupling between phosphate release and force generation in muscle actomyosin
Phil. Trans. Roy. Soc. ser. B
The mechanism of muscular contraction
Science
Studies on the “hinge” region of myosin
Biochemistry
Melting of myosin rod as revealed by electron microscopy. II. Effects of temperature and pH on length and stability of myosin rod and its fragments
Eur. J. Cell Biol.
On the origin of the contractile force in skeletal muscle
Proc. Natl Acad. Sci. USA
Conformational transition in the myosin hinge upon activation of muscle
Proc. Natl Acad. Sci. USA
Effects of ions and pH on the thermal stability of thin and thick filaments of skeletal muscle: high-sensitivity differential scanning calorimetric study
Biochemistry
Influence of the cardiac myosin hinge region on contractile activity
Proc. Natl Acad. Sci. USA
Contraction characteristics and ATPase activity of skeletal muscle fibers in the presence of antibody to myosin subfragment 2
Proc. Natl Acad. Sci. USA
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Present address: C. M. Dambacher, The Scripps Research Institute, Kellogg School of Science and Technology, 10550 N. Torrey Pines Road, TPC-19, La Jolla, CA 92037, USA.