Fifty Ways to Love Your Lever: Myosin Motors
1996; Cell Press; Volume: 87; Issue: 2 Linguagem: Inglês
10.1016/s0092-8674(00)81332-x
ISSN1097-4172
Autores Tópico(s)Genetic Neurodegenerative Diseases
ResumoThe ability to produce directed motion is a distinguishing characteristic of practically all living organisms. Over a century has passed since Kühne first extracted the proteins actin and myosin from muscle (41Squire J. The Structural Basis of Muscle Contraction. Plenum Press, New York1981Crossref Google Scholar), yet the molecular origin of the force produced between these two components remains one of the outstanding puzzles in biology. Progress towards an understanding of how muscles develop force was made in the middle of this century with the development of the sliding filament model, immortalized today in undergraduate cell biology textbooks (for historical perspective, see14Huxley A.F. Reflections on Muscle.in: The Sherrington Lectures XIV. Princeton University Press, Princeton, New Jersey1980Google Scholar, 17Huxley H.E. A personal view of muscle and motility mechanisms.Ann. Rev. Physiol. 1996; 58: 1-19Crossref PubMed Scopus (44) Google Scholar). It is now well-established that molecules of the ATPase myosin bind to, and slide along, filaments of actin. In fact, direct visualization of this motion at the macromolecular level is possible today, using in vitro motility assays consisting of purified components (33Scholey, J. (ed.) (1993). Motility Assays for Motor Proteins. In: Meth. Cell Biology V. 39 (New York: Academic Press).Google Scholar). The force produced by actomyosin is not only harnessed on a grand scale in muscle, but also underlies a host of microscopic motions, including cell motility, cytokinesis, vesicle transport, and cellular shape changes. Along with myosin, other linear motor families have since been identified, including dynein and kinesin, which move along microtubules. There is reason to believe that the molecular mechanism of these other motor proteins may, at the end of the day, resemble that of myosin. But what physical and chemical changes hold the key to the action of motor proteins? And how is the hydrolysis of ATP thereby coupled to motion? These fundamental questions have proved very hard to answer. Over the decades, the motility problem has spawned endless debates and countless numbers of competing models. A vast literature of biochemical and biophysical data has been amassed, particularly for myosin, which is arguably the best-characterized of proteins (41Squire J. The Structural Basis of Muscle Contraction. Plenum Press, New York1981Crossref Google Scholar, 2Bagshaw C.R. Muscle Contraction. 2. Chapman and Hall, London1993Crossref Google Scholar). Despite the wealth of information, the fundamental questions remain. Testifying to the ongoing controversy, there is not even consensus about whether movement is powered by changes taking place primarily in the myosin head — the conventional dogma — or by shape changes within the actin filament itself (e.g.,34Schutt C.E. Lindberg U. Actin as the generator of tension during muscle contraction.Proc. Natl. Acad. Sci. USA. 1992; 89: 319-323Crossref PubMed Scopus (58) Google Scholar), and both alternatives remain formally possible. Despite this, many investigators hold to the view that it is the myosin head that undergoes some kind of conformational change, or "power stroke", causing it to step forward cyclically along the actin, ratcheting in the direction of motion. This notion emerged from seminal work on muscle fibers (16Huxley H.E. The mechanism of muscle contraction.Science. 1969; 164: 1356-1366Crossref PubMed Scopus (1270) Google Scholar, 15Huxley A.F. Simmons R.M. Proposed mechanism of force generation in striated muscle.Nature. 1971; 233: 533-538Crossref PubMed Scopus (1562) Google Scholar) and led to the suggestion that myosin might produce a power stroke of around 12 nm: an enormous distance for a protein, even one as big as myosin. One way to leverage up the power stroke would be for the entire myosin head to rock as an entity about its point(s) of contact with the actin filament: the swinging crossbridge model. Throughout the 1970's and 80's, sophisticated biophysical techniques were used to hunt for signs of crossbridge rotation, including EPR and fluorescence spectroscopy, X-ray and neutron scattering, electric birefringence, etc. Much of the evidence was equivocal, at best. For example, spin probes attached to the primary reactive thiol of the myosin head (known as SH-1) reported little, if any, net angular movement during active muscle contraction, as compared to at rest (reviewed by12Highsmith S. Cooke R. Evidence for actomyosin conformational changes in tension generation.J. Cell Res. Muscle Motil. 1983; 4: 207-237Crossref PubMed Scopus (5) Google Scholar, 7Cooke R. The mechanism of muscle contraction.C.R.C. Crit. Rev. Biochem. 1986; 21: 53-118Crossref PubMed Scopus (232) Google Scholar). These sorts of negative result led to the salvage proposal that the bulk of the head might not rotate after all, but that its more distal tail portion (i.e., the remainder of the S1 proteolytic head fragment) wagged nevertheless (7Cooke R. The mechanism of muscle contraction.C.R.C. Crit. Rev. Biochem. 1986; 21: 53-118Crossref PubMed Scopus (232) Google Scholar). During the first half of this decade, two significant breakthroughs occurred in diverse areas of biophysics. These advances, in conjunction with established methods in molecular biology, have renewed hope that a resolution of the myosin problem might be close at hand, perhaps by the dawn of the coming millennium. First, the atomic structures for both the actin monomer (21Kabsch W. Mannherz H.-G. Suck D. Pai E.F. Holmes K.C. Atomic structure of the actin:DNase I complex.Nature. 1990; 347: 37-44Crossref PubMed Scopus (1491) Google Scholar, 35Schutt C.E. Myslik J.C. Rozycki M.D. Gooneesekere N.C.W. Lindberg U. The struture of profilin:β-actin.Nature. 1993; 365: 810-816Crossref PubMed Scopus (585) Google Scholar, 25McLaughlin P.J. Gooch J.T. Mannherz H.-G. Weeds A.G. Structure of gelsolin segment 1-actin complex and the mechanism of filament severing.Nature. 1993; 364: 685-692Crossref PubMed Scopus (488) Google Scholar) and the S1 head fragment of myosin (30Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Three-dimensional structure of myosin subfragment-1 a molecular motor.Science. 1993; 261: 50-58Crossref PubMed Scopus (1815) Google Scholar) were solved, in a tour de force of X-ray diffraction. Both proteins had defied crystallographers for decades. Armed with high-resolution structural data for the monomers, it became possible to combine this information with lower-resolution data, obtained by X-ray diffraction of actin fibers or electron microscope-based reconstructions of actomyosin complexes, and thereby formulate atomic-level models for the actin filament (13Holmes K.C. Popp D. Gebhard W. Kabsch W. Atomic model of the actin filament.Nature. 1990; 347: 44-49Crossref PubMed Scopus (1286) Google Scholar) and the actin filament decorated by myosin heads bound in rigor (a reference to the Latin rigor mortis, the ATP-depleted state that frequently follows death) (31Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Structure of the actin-myosin complex and its implications for muscle contraction.Science. 1993; 261: 58-65Crossref PubMed Scopus (1415) Google Scholar, 36Schröder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1.Nature. 1993; 364: 171-174Crossref PubMed Scopus (265) Google Scholar). Here, at last, was a plausible picture of actomyosin during at least part of its mechanochemical cycle. The second breakthrough occurred when in vitro motility assays were successfully married with ultrasensitive optical instrumentation, capable of recording both force and displacements down to the molecular level, all in the light microscope. This made it possible for the first time to measure directly the steps taken by individual motor molecules, such as kinesin or myosin. For this purpose, laser-based optical traps ("optical tweezers,"42Svoboda K. Schmidt C.F. Schnapp B.J. Block S.M. Direct observation of kinesin stepping by optical trapping interferometry.Nature. 1993; 365: 721-727Crossref PubMed Scopus (1528) Google Scholar; Finer et al., 1994; 27Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C.S. Movement and force generated by a single myosin head.Nature. 1995; 378: 209-212Crossref PubMed Scopus (508) Google Scholar) and fine glass microneedles have been used (19Ishijima A. Harada Y. Kojima H. Funatsu T. Higuchi H. Yanagida T. Biochem. Biophys. Res. Commun. 1994; 199: 1057-1063Crossref PubMed Scopus (173) Google Scholar). Single myosin interactions have been scored whose mean displacements range from 5-25 nm (with forces developed of 1-5 pN). However, a key point of controversy remains as to whether these individual mechanical events correspond to a single ATP hydrolysis, or whether one ATP might somehow lead to multiple steps (49Yanagida T. Harada Y. Ishijima A. Nano-manipulation of actomyosin molecular motors in vitro a new working principle.Trends in Biochem Sci. 1993; 18: 319-324Abstract Full Text PDF PubMed Scopus (46) Google Scholar; Finer et al., 1994). This question lies at the heart of issues about mechanochemical coupling (5Burton K. Myosin step size estimates from motility assays and shortening muscle.J. Muscle Res. and Cell Motil. 1992; 13: 590-607Crossref PubMed Scopus (41) Google Scholar). The S1 crystal structure and the corresponding model for myosin bound to actin led Rayment and colleagues to propose a conformational change-based model that bears unmistakable similarities to the tail-wagging idea which represented the fallback position at the end of the 1980's (31Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Structure of the actin-myosin complex and its implications for muscle contraction.Science. 1993; 261: 58-65Crossref PubMed Scopus (1415) Google Scholar). This time around, however, the model was a bit more specific and had a firm basis in structure that could lead, in principle, to definitive tests of functional relationships. Proteolytic susceptibility had long ago been used to identify three distinct fragments of the S1 heavy chain polypeptide, named for their sizes: the 50 kDa, 25 kDa, and 20 kDa regions (Figure 1). Actin binding is mediated by the 50 kDa domain, the ATPase site spans the 50-25 kDa domain interface, and the 20 kDa domain binds the two light chains. A striking feature of the crystal structure is that the 20 kDa domain consists almost entirely of an exceptionally long, uninterrupted α helix, comprising 70+ amino acids, that is presumably prevented from spontaneous collapse (i.e., rigidified) by its interactions with the two light chains, which envelop it along most of its ∼9 nm length. The immediate possibility suggested by this feature is that it might somehow serve as a "lever arm" to drive the rest of the molecule forward when rotated at its base through some hydrolysis-induced angle (see40Spudich J.A. How molecular motors work.Nature. 1994; 372: 515-518Crossref PubMed Scopus (414) Google Scholar). Clearly, such a mechanism could mechanically amplify smaller motions in the head. But can it explain molecular steps believed to be ∼10 nm, perhaps greater? And are such large-amplitude motions, in fact, required? Without substantial rearrangements, the crystal structure only seems to admit to motions of 5 or 6 nm (31Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Structure of the actin-myosin complex and its implications for muscle contraction.Science. 1993; 261: 58-65Crossref PubMed Scopus (1415) Google Scholar), and at least one report of myosin step size falls within this range (27Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C.S. Movement and force generated by a single myosin head.Nature. 1995; 378: 209-212Crossref PubMed Scopus (508) Google Scholar).Figure 1Hypothetical Model of the Swinging Lever ArmShow full caption(A) A computer-based visualization of the prestroke complex. Right, running vertically: a ribbon representation of a portion of an actin filament, positioned according to the model of (Lorenz et al. 1993), showing five identical monomers colored either slate blue or light grey. The barbed end of the filament is towards the bottom; myosin moves along the direction shown (arrow). Left: A ribbon representation of a single chicken myosin S1 head bound to actin, based on Rayment et al., 1993b. The color convention follows (Rayment, et al. 1993a): magenta, regulatory light chain (RLC); yellow, essential light chain (ELC); green, heavy chain 25 kDa domain (amino acids 4-217); red, heavy chain 50 kDa domain (amino acids 218-625); dark blue, heavy chain 20 kDa domain (amino acids) 648-843). The α helices of the 20 kDa subunit bearing reactive thiols SH-1 and SH-2 have been colored cyan. A short loop (amino acids 626-647) at the junction of the 50 kDa and 20 kDa domains, unresolved in the chicken crystal structure, has been colored white: this loop has been implicated in control of the ATPase cycling rate (Spudich 1994). A part of the bound nucleotide (grey) can be seen in the cleft between the 25 and 50 kDa domains, just to the right of the 20 kDa domain. To model the conformational change, the myosin structure has been deliberately altered, rotating the long helical portion of the 20 kDa domain and associated light chains about a pivot near the base of the lever arm, to depict what this complex might resemble prior to the power stroke. The 50 kDa and 25 kDa domains have not been altered, and molecular collisions occur in the model: rearrangements are anticipated for these regions as well, particularly near the SH1-SH2 helices (see text).(B) The post-stroke state. This panel depicts the actomyosin complex in a rigor-like configuration, as in (Rayment et al. 1993b). It is assumed here, as in previous work, that the myosin S1 crystal structure has the same shape as in rigor (no bound nucleotide), and also that the rigor configuration displays a similar orientation with respect to actin as that following the power stroke, prior to ADP release. A rotation of the "lever arm" through ∼90° would produce a step of ∼12 nm, thereby pulling the remainder of the myosin molecule (not shown) downwards. Smaller steps would correspond to less severe rotation. Composition and color color scheme are identical to (A). Models in (A) and (B) were created by K. C. Holmes using GRASP software.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) A computer-based visualization of the prestroke complex. Right, running vertically: a ribbon representation of a portion of an actin filament, positioned according to the model of (Lorenz et al. 1993), showing five identical monomers colored either slate blue or light grey. The barbed end of the filament is towards the bottom; myosin moves along the direction shown (arrow). Left: A ribbon representation of a single chicken myosin S1 head bound to actin, based on Rayment et al., 1993b. The color convention follows (Rayment, et al. 1993a): magenta, regulatory light chain (RLC); yellow, essential light chain (ELC); green, heavy chain 25 kDa domain (amino acids 4-217); red, heavy chain 50 kDa domain (amino acids 218-625); dark blue, heavy chain 20 kDa domain (amino acids) 648-843). The α helices of the 20 kDa subunit bearing reactive thiols SH-1 and SH-2 have been colored cyan. A short loop (amino acids 626-647) at the junction of the 50 kDa and 20 kDa domains, unresolved in the chicken crystal structure, has been colored white: this loop has been implicated in control of the ATPase cycling rate (Spudich 1994). A part of the bound nucleotide (grey) can be seen in the cleft between the 25 and 50 kDa domains, just to the right of the 20 kDa domain. To model the conformational change, the myosin structure has been deliberately altered, rotating the long helical portion of the 20 kDa domain and associated light chains about a pivot near the base of the lever arm, to depict what this complex might resemble prior to the power stroke. The 50 kDa and 25 kDa domains have not been altered, and molecular collisions occur in the model: rearrangements are anticipated for these regions as well, particularly near the SH1-SH2 helices (see text). (B) The post-stroke state. This panel depicts the actomyosin complex in a rigor-like configuration, as in (Rayment et al. 1993b). It is assumed here, as in previous work, that the myosin S1 crystal structure has the same shape as in rigor (no bound nucleotide), and also that the rigor configuration displays a similar orientation with respect to actin as that following the power stroke, prior to ADP release. A rotation of the "lever arm" through ∼90° would produce a step of ∼12 nm, thereby pulling the remainder of the myosin molecule (not shown) downwards. Smaller steps would correspond to less severe rotation. Composition and color color scheme are identical to (A). Models in (A) and (B) were created by K. C. Holmes using GRASP software. The original structure of chicken myosin S1 had sulfate, as opposed to ATP or ADP, in the enzyme active site. This raised the question whether the crystallized form reflected the shape of the native protein before, or after, the hydrolytic event postulated to produce conformational changes—or perhaps something else again. Put simply, would myosin crystallized with different substrates have different shapes? To address this question, Rayment's group has crystallized and solved a series of shorter myosin heavy chain fragments from Dictyostelium with various bound nucleotide and transition-state analogs, including Mg.ADP.BeFx, Mg.ADP.AlF4−, Mg.ADP.vanadate, and Mg.PPi (10Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. X-ray structures of the myosin motor domain of Dictyostelium complexed with MgADP.BeFx and MgADP.AlF4-.Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (621) Google Scholar, 38Smith C.A. Rayment I. X-ray structure of the magnesium(II)-pyrophosphate complex of the truncated head of Dictyostelium discoideum myosin to 2.7 Å resolution.Biochemistry. 1995; 34: 8973-8981Crossref PubMed Scopus (89) Google Scholar, 39Smith C.A. Rayment I. X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 Å resolution.Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (505) Google Scholar). To obtain these crystals, it was necessary to work with protein fragments too abbreviated to carry the light chains (∼730-740 amino acids), so positions of the "lever arm" were not determined. Broadly speaking, the results fall into two classes. Structures with bound ADP-beryllium fluoride, Mg-pyrophosphate, and sulfate are similar to one another, and would seem to correspond to an "ATP-like", prehydrolysis form. The structures with bound ADP-aluminum fluoride or ADP-vanadate form a second class. These are again similar to one another, but display various structural changes distinct from the first class, and would seem to be candidates for a "transition-state" form. Although large-scale structural changes were seen in certain "transition-state" structures near the carboxy-end of the structures where the lever arm would emerge, these occur in a part of the molecule that may not be structurally trustworthy, by virtue of the polypeptide being unnaturally lopped off near that point for crystallization purposes. It seems fair to say that the lever arm hypothesis has not yet been corroborated by crystallographic work, although there are tantalizing hints in the structural data that a subdomain of the molecule near the 25 kDa–20 kDa interface, optimistically dubbed by some the "converter" region, might undergo substantial changes. There is ample reason to believe that major rearrangements must take place during the myosin mechanochemical cycle. There are two reactive sulfhydryl groups located on cysteines in skeletal muscle S1, designated as SH-1 and SH-2. These sulfhydryls (Cys707 and Cys697, respectively) are found on consecutive regions of α helix joined by a short turn, and spatially separated in the chicken myosin structure by ∼1.8 nm (31Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Structure of the actin-myosin complex and its implications for muscle contraction.Science. 1993; 261: 58-65Crossref PubMed Scopus (1415) Google Scholar). However, they can be crosslinked by a variety of bifunctional reagents that span distances as short as 0.3 nm (4Burke M. Reisler E. Effect of nucleotide binding on the proximity of the essential sufhydryl groups of myosin chemical probing of movement of residues during conformational transitions.Biochem. 1977; 16: 5559-5563Crossref PubMed Scopus (85) Google Scholar). Moreover, crosslinking of SH-1 and SH-2 results in the trapping of nucleotide, in ADP form, at the active site of the enzyme (47Wells J.A. Yount R.G. Active site trapping of nucleotides by crosslinking two sufhydryls in myosin subfragment 1.Proc. Natl. Acad. Sci. USA. 1979; 76: 4966-4970Crossref PubMed Scopus (121) Google Scholar). Clearly, some structural alteration must occur concomitant with hydrolysis that shortens the distance from SH-1 to SH-2: one not yet reported for the crystal structures. Indeed, structural evidence for certain large-scale motions of myosin S1 has emerged recently, but this has raised more questions than it answers. Actin filaments decorated with either the S1 fragment of smooth muscle myosin II or brush border myosin I were bathed in solutions containing high levels of MgADP, to generate complexes containing the ADP-bound form, as opposed to the nucleotide-free form of rigor (48Whittaker M. Wilson-Kubalek E.M. Smith J.E. Faust L. Milligan R.A. Sweeney H.L. A 35-Å movement of smooth muscle myosin on ADP release.Nature. 1995; 378: 748-751Crossref PubMed Scopus (335) Google Scholar, 20Jontes J.D. Wilson-Kubalek E.M. Milligan R.A. A 32° tail swing in brush border myosin I on ADP release.Nature. 1995; 378: 751-753Crossref PubMed Scopus (165) Google Scholar). 3D cryoelectron microscope reconstructions of such filaments showed heads bound with the characteristic "arrowhead" pattern seen for skeletal muscle myosin. In the main, the head shapes were roughly similar to those previously observed with skeletal muscle myosin in rigor (26Milligan R.A. Flicker P.F. Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy.J. Cell Biol. 1987; 105: 29-39Crossref PubMed Scopus (200) Google Scholar), but with a twist: the tail portions of these molecules had undergone extensive rotations with respect to the rigor forms: ∼23° for smooth muscle S1 (corresponding to a displacement of ∼3.5 nm at the end of the tail) and ∼35° for brush border myosin I (corresponding to a displacement of 5.0 to 7.2 nm at the end of the tail). Could this be the smoking gun? Probably not. Conventional models of force generation (40Spudich J.A. How molecular motors work.Nature. 1994; 372: 515-518Crossref PubMed Scopus (414) Google Scholar) don't place the power stroke in the part of the cycle corresponding to ADP release. Also, the free energy change associated with ADP release is rather small (although the large energy drops elsewhere in the sequence might suffice, in principle, given the cyclical nature of the reaction scheme). Finally, these changes simply are not seen in skeletal myosin. In follow-up work with EPR spectroscopy, Cooke and colleagues placed spin probes on the regulatory light chain of smooth muscle myosin. When the labeled chains were exchanged for native ones, changes in the mean angles of probe orientation of up to 20° were found in muscle upon addition of ADP. Conversely, similar experiments with skeletal muscle myosin failed to produce any significant change in the mean orientation angle (11Gollub J. Cremo C.R. Cooke R. ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin.Nature Struct. Biol. 1996; 3: 796-802Crossref PubMed Scopus (74) Google Scholar). If not a power stroke, to what, then, does the ADP-induced shape correspond? Milligan, Sweeney, and colleagues speculate that it might be the so-called "latch-bridge" state, which is the smooth muscle analog of the catch-bridge state of molluscan myosins, whereby muscle fibers are able to lock up in contracted forms and sustain loads without a continual need to burn ATP (48Whittaker M. Wilson-Kubalek E.M. Smith J.E. Faust L. Milligan R.A. Sweeney H.L. A 35-Å movement of smooth muscle myosin on ADP release.Nature. 1995; 378: 748-751Crossref PubMed Scopus (335) Google Scholar). Assuming this interpretation is correct, it raises the specter that there may be a multitude of structural forms associated with the mechanochemical cycle. A collaboration among several labs in the U.S. and U.K. has used fluorescence polarization spectroscopy to identify orientational changes of the light chains during muscle movement. Chicken gizzard light chains were expressed in E. coli, labeled with a single reactive rhodamine fluorophore at Cys108, and exchanged into rabbit skeletal muscle (18Irving M. St. Claire Allen T. Sabido-David C. Craik J.S. Brandmeier B. Kendrick-Jones J. Corrie J.E.T. Trentham D.R. Goldman Y.E. Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle.Nature. 1995; 375: 688-691Crossref PubMed Scopus (188) Google Scholar). Measurements of polarization states at rest, during active muscle contraction, and under stretch were consistent with a tilting of the light chain "lever arm" region. However, the inferred angular change was disappointingly small: just ∼3°, even assuming that all probes in the ordered fraction responded. One explanation might be that the real angular change is much larger, but that only a tiny fraction of heads in the muscle fiber bear force and respond to length steps, and there is some support for this view from in vitro studies. The same group is now attaching fluorescence probes that bind to two reactive thiols and thereby cannot rotate about the attachment point, resolving angular ambiguities inherent in the original approach. The use of two or more such light chain probes oriented (nearly) orthogonally to one another should provide unprecedented resolution of molecular changes in real time. If the 20 kDa region truly functions as a kind of lever arm, then changes in the lever arm length might produce corresponding changes in the myosin step size. This line of thinking has been pursued actively by Spudich and coworkers, who genetically engineered mutant Dictyostelium myosins with different sizes of lever arm, altered by changing the number of light chain binding regions (45Uyeda T.Q.P. Abramson P.D. Spudich J.A. The neck region of the myosin motor domain acts as a lever arm to generate movement.Proc. Natl. Acad. Sci. USA. 1996; 93: 4459-4464Crossref PubMed Scopus (373) Google Scholar). Three variants were created. The first was deleted for both light chain binding sites, the second was deleted for the regulatory chain binding site, while the third carried a tandem repeat of the essential light chain binding site along with the normal regulatory site, endowing it with three light chains. The three mutant constructs, together with the wild type and its twin light chain binding sites, constitute a series with 0, 1, 2, or 3 light chains of increasing length. The four proteins were expressed in cells, purified, and scored for motility in vitro and for ATPase activity. All four moved actin in vitro, at average sliding velocities that were found to increase monotonically with the number of binding sites. Not only did the shorter lever arm constructs move correspondingly slowly, but importantly, the one with an additional light chain site moved even faster than the wild type. In fact, the sliding velocities were in strict linear proportion to the lengths of the putative lever arms: a result almost too good to be true! On the assumption that the sliding velocity is proportional to the step size, this linear relationship permits the data from Dictyostelium to be extrapolated back into the (nearly identical) chicken myosin structure to locate the approximate fulcrum point of the lever, which turned out to be at the very base of the 20 kDa region, near the location of the α helices bearing the reactive thiols SH-1 and SH-2. The underlying assumption in this work is that the sliding velocity of filaments in vitro, v, identically reflects the myosin step size, d. This will only be true when the step timing is exactly the same for each of the different myosins, since v = d/τ, with τ being something like the time taken per step. Unfortunately, τ as just described is ill-defined. Does one take for τ the time required for a complete ATPase cycle (i.e., the reciprocal of the turnover rate)? If so, then the "lever arm interpretation" of the experiment fails, since ATPase rates for the constructs differ from wild type by factors of ∼2. Spudich and company argued, with some justification, that the relevant time to consider is not the turnover time, but rather a time corresponding to that fraction of the cycle during which myosin and actin are tightly bound and can develop force, i.e., the strong-binding time, τs. This time is significantly shorter than the overall cycle time, occupying ∼5% or less of the cycle in wild type. But, then, are the strong-binding times identical in wild type and all the mutant constructs? That remains to be demonstrated. A
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