Kinetic Analysis of Drosophila Muscle Myosin Isoforms Suggests a Novel Mode of Mechanochemical Coupling

Abstract

The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by ␣-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin⅐myosin⅐ADP states may define actin filament velocity for these myosin isoforms.Cyclical shortening and lengthening of a muscle fiber is the direct result of myosin cross-bridges transiently interacting with actin filaments utilizing the energy from ATP hydrolysis. Specific muscle fibers show characteristic biochemical and mechanical properties that are largely governed by the isoform of myosin expressed (1, 2). It is widely accepted that the overall cross-bridge cycle of muscle myosin is conserved; thus alterations in this cycle are the result of changes in the biochemical kinetic constants of individual events resulting directly from myosin sequence changes. Recent work supports the inference that in Drosophila muscle myosin isoform functional diversity is the result of kinetic differences governing strong binding attachment and/or detachment kinetics rather than changes in step size (3-5). Structural studies have focused on identifying regions of the myosin molecule, in both muscle and non-muscle isoforms, containing naturally occurring variations that could account for these isoform-specific properties (6 -11). Functional studies of these natural variants along with intentionally modified myosin isoforms have begun to probe the structure-function relationship critical to elucidating the molecular mechanism of myosin (for reviews, see Refs. 12 and 13).To study specific regions and amino acids within myosin that define the complex structure-function relationship, we have turned to the genetically tractable organism, Drosophila melanogaster. This model system allows the creation of transgenic organisms containing altered regions or amino acids within myosin to probe the functional contribution of these residues in the mechanochemical cycle (14). Recent studies have shown that Drosophila muscle-specific myosin isoform properties are deter...

An exceptionally fast actomyosin reaction powers insect flight muscle

“…At saturating levels of MgATP (10-20 mM), the IFI data uniquely fit a scheme in which Pi release is the rate-limiting step ( Fig. 3 A and C), in accord with a recent biochemical study that suggests MgADP release is extremely fast from Drosophila IFI myosin (15). The slow EMB-fiber data, on the other hand, fit a configuration in which the rate-limiting step is an isomerization of myosin before MgADP release ( Fig.…”

“…2 E and F, we conclude that MgADP release must be very fast (and therefore K ADP very low) to achieve a forward detachment rate constant k 2 of 3,698 s Ϫ1 for IFI at 15°C (Table 1). Further evidence for a very fast MgADP release rate from IFI comes from solution studies of IFI myosin, where an off rate of 4,090 s Ϫ1 was calculated based on a measured MgADP-affinity constant of 2.5 mM Ϫ1 (at 22°C) (15). MgADP release is likely to be even faster in fibers because of the effects of mechanical stress and͞or strain on the myosin head (see Supporting Methods for calculated estimate).…”

“…In most vertebrate striated muscle types, the comparatively slow release of MgADP (one of the products of MgATP hydrolysis) is thought to be the ratelimiting step (8)(9)(10)(11)(12)(13). However, recent studies suggest MgADP release may not be rate limiting for faster muscle types (14)(15)(16). If so, we reasoned that a shift in rate-limiting step to another part of the cross-bridge cycle should be most readily apparent in working indirect insect flight muscle, the fastest known muscle type.…”

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

“…Correspondingly, the crystal structure of chicken skeletal S1 in the near-rigor state (4) displays the same hydrogen-bonded and arginine-mediated complex salt bridge link between switch I and the N-terminal subdomain as observed in the present structures. Moreover, a recent study (38) places the Drosophila embryonic and indirect flight muscle myosins in the high-coupling category, and sequence alignment reveals that the corresponding residues in the Drosophila muscle myosins are again identical to those in scallop (Table 2). By contrast, the chicken smooth muscle MD prepower-stroke state crystal structure (8) reveals only a weak, lysine-mediated simple salt bridge in this region (Table 2).…”

Nature’s Strategy for Optimizing Power Generation in Insect Flight Muscle

“…Conversely, fibers expressing the chimera made by the opposite exchange (replacing the native converter with the EMB converter) reduced P by a third and f by one half compared to native IFM (i.e., IFI-EC vs IFI: Table 1) . Preliminary experiments suggest other variable regions do not play as great a role in determining myofibril kinetics (Miller et al, 2003;Swank et al, 2002a;Swank et al, 2004), supporting the hypothesis that the converter region is a strategic location.…”