A closer look at energy transduction in muscle.

Muscular force is the sum of unitary force interactions generated as filaments of myosins move forcibly along parallel filaments of actins, understanding that the free energy required comes from myosin-catalyzed ATP hydrolysis. Using results from conventional biochemistry, our own mutational studies, and diffraction images from others, we attempt, in molecular detail, an account of a unitary interaction, i.e., what happens after a traveling myosin head, bearing an ADP-P(i), reaches the next station of an actin filament in its path. We first construct a reasonable model of the myosin head and actin regions that meet to form the "weakly bound state". Separately, we consider Holmes' model of the rigor state [Holmes, K. C., Angert, I., Kull, F. J., Jahn, W. & Schröder, R. R. (2003) Nature 425, 423-427], supplemented with several heretofore missing residues, thus realizing the "strongly bound state." Comparing states suggests how influences initiated at the interface travel elsewhere in myosin to discharge various functions, including striking the actins. Overall, state change seems to occur by attachment of a hydrophobic triplet (Trp-546, Phe-547, and Pro-548) of myosin to an actin conduit with a hydrophobic guiding rail (Ile-341, Ile-345, Leu-349, and Phe-352) and the subsequent linear movement of the triplet along the rail.

Toward understanding actin activation of myosin ATPase: the role of myosin surface loops.

To understand the complicated interplay when a traveling myosin head reaches interaction distance with two actins in a filament we looked to three myosin loops that early on exert their influences from the "outside" of the myosin. On these we conduct, functionally test, and interpret strategically chosen mutations at sites thought from crystallography to be a patch for binding the "first" of the two actins. One loop bears a hydrophobic triplet of residues, one is the so-called "loop 2," and the third is the "cardiomyopathy" loop. So far as we know, the myosin sites that first respond are the two lysine-rich loops that produce an ionic strength-dependent weak-binding complex with actin. Subsequently, the three loops of interest bind the first actin simultaneously, and all three assist in closing the cleft in the 50-kDa domain of the myosin, a closure that results in transition from weak to strong binding and precedes rapid Pi release and motility. Mutational analysis shows that each such loop contact is distinctive in the route by which it communicates with its specific target elsewhere in myosin. The strongest contact with actin, for example, is that of the triplet-bearing loop. On the other hand, that of loop 2 (dependent on drawing close two myosin lysines and two actin aspartates) is probably responsible for opening switch I and uncovering the gamma-phosphate moiety of bound ATP. Taking into account these findings, we begin to arrange in order many molecular events in muscle function.

Transmission of force and displacement within the myosin molecule.

Myosin is a repetitive impeller of actin, using its catalysis of ATP hydrolysis to derive repeatedly the required free energy decrements. In each impulsion, changes at the myosin active site are transmitted through a series of structural elements to the myosin propeller (lever arm), almost 5 nm away. While the nature of transmission through most elements is evident, that through the so-called converter is not. To investigate how the converter changes linear displacement into rotation, we tested (one at a time) the effect of two Phe residue mutations (at 721 and 775) in the converter on the overall function of a heavy meromyosin (or subfragment 1) system, after first showing by observing kinetic behaviors that neither mutation affects other elements in the transmission. Using three tests (direct movement of the lever arm, activity in a motility assay with actin filaments, and direct force measurement of lever arm function), we found that these mutations affected only movements of the converter and the lever arm. From interpreting our observations in terms of the structure of the converter, we deduce that the linear-rotational transformation in the converter is mediated by a little machine (two Phe residues linked to a Gly) within a machine.

On the myosin catalysis of ATP hydrolysis.

Myosin is an ATP-hydrolyzing motor that is critical in muscle contraction. It is well established that in the hydrolysis that it catalyzes a water molecule attacks the gamma-phosphate of an ATP bound to its active site, but the details of these events have remained obscure. This is mainly because crystallographic search has not located an obvious catalytic base near the vulnerable phosphate. Here we suggest a means whereby this dilemma is probably overcome. It has been shown [Fisher, A. J., et al. (1995) Biochemistry 34, 8960-8972; Smith, C. A., and Rayment, I. (1996) Biochemistry 35, 5404-5417] that in an early event, Arg-247 and Glu-470 come together into a "salt-bridge". We suggest that in doing so they also position and orient two contiguous water molecules; one of these becomes the lytic water, perfectly poised to attack the bound gamma-phosphorus. Its hydroxyl moiety attacks the phosphorus, and the resulting proton transfers to the second water, converting it into a hydronium ion (as is experimentally observed). It is shown in this article how these central events of the catalysis are consistent with the behavior of several residues of the neighboring region.

A hypothesis about myosin catalysis.

When ATP binds to the active site of myosin heads, Switch II undergoes a large conformational change and the cleft surrounding the bound gamma-phosphate closes. In the closed state, Glu470 in Switch II comes together with Arg247 in Switch I to form a salt-bridge. Here, the functional significance of the two bridging residues was tested by using site-directed mutagenesis. We conclude from such tests that (a) the attractive force between Arg247 and the gamma-phosphate of ATP moves the cleft to close, and (b) during hydrolysis, Glu470 is intimately involved in positioning the lytic water for the attack on the gamma-phosphorus. We also speculate on how the salt-bridge between Arg247 and Glu470 is related to hydrolysis.

Early stages of energy transduction by myosin: roles of Arg in switch I, of Glu in switch II, and of the salt-bridge between them.

On the basis of the crystallographic snapshots of Rayment and his collaborators [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960-8972], we have understood some basic principles about the early stages of myosin catalysis, namely, ATP is drawn into the active site, over which the cleft closes. Catalyzed hydrolysis occurs, and the first product (orthophosphate) is released from the backdoor of the cleft. In the cleft-closing process, the active site incidentally signals its movement to a particular remote tryptophan residue, Trp-512. In this work, we expand on some of these ideas to rationalize the behavior of a mutated system in action. From the behavior of recombinant myosin systems in which Arg-247 and Glu-470 were substituted in several ways, we draw the conclusions that (i) the force between Arg-247 and gamma-phosphate of ATP may assist in closing the cleft, and incidentally in signaling to the remote Trp, and (ii) in catalysis, Glu-470 is involved in holding the lytic H(2)O (w(1)). We also propose that w(1) and also a second water, w(2), enter into a structure that bridges Glu-470 and the gamma-phosphate of bound ATP, and at the same time positions w(1) for its in-line hydrolytic attack.

Changes in the 31P NMR spectrum of rabbit muscle myosin subfragment 1. MgADP with temperature.

In pioneering studies on the 31P NMR spectra of MgADP bound to the "molecular motor" myosin subfragment 1 (S1) in the temperature range of 0 to 25 degrees C, Shriver and Sykes [Biochemistry 20 (1981) 2004-2012/6357-6362; Biochemistry 21 (1982) 3022-3028], proposed that MgADP binds to myosin S1 as a mixture of two interconvertible conformers with different chemical shifts for the beta-P resonance of the S1-bound MgADP and that the concentrations of these conformers are related by an equilibrium constant K(T). Their model implied that the weighted average of the chemical shifts of the beta-P(MgADP) for S1-bound MgADP asymptotically approaches a high temperature limit. Here, and in our earlier paper [K. Konno, K. Ue, M. Khoroshev, H., Martinez, B.D. Ray, M.F. Morales, Proc. Natl. Acad. Sci. USA 97 (2000) 1461-1466], we report experimental similarities to Shriver and Sykes, but diverge from them (especially at 0 degrees C) in not finding two distinct peaks and in finding that the average chemical shift does not change with temperature. Our observations can be explained by chemical exchange of beta-P(MgADP) of S1-bound MgADP between two nearly energetically equivalent environments.