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(The FASEB Journal. 2004;18:783-789.)
© 2004 FASEB

Is peptide bond cis/trans isomerization a key stage in the chemo-mechanical cycle of motor proteins?

OREN TCHAICHEEYAN1

Biomedical Engineering Department, Technion - Israel Institute of Technology, Haifa, Israel

1Correspondence: Biomedical Engineering Department, Technion - Israel Institute of Technology, Haifa, 32000, Israel. E-mail: toren{at}tx.technion.ac.il

ABSTRACT

Motor proteins such as myosin and kinesin are responsible for actively directed movement in vivo. The physicochemical mechanism underlying their function is still obscure. A novel and unifying model concerning the motors driving mechanism is suggested here. This model resides within the framework of the well-studied "swinging lever-arm" hypothesis, stating that cis/trans peptide bond isomerization (CTI) is a key stage in the chemo-mechanical coupling within actomyosin—the complex of the motor (myosin) and its specific track (actin). CTI is suggested to propel myosin’s lever-arm swing. The model addresses on the submolecular level a broad spectrum of actomyosin’s functional characteristics, such as kinetics, energetics, force exertion, stepping, and directionality. The model may be tested first with relative ease in kinesin—a smaller motor that could be specifically modified with unnatural amino acids using bacterial expression. Suggested modifications may be used for labeling and functional decoupling.—Tchaicheeyan, O. Is peptide bond cis/trans isomerization a key stage in the chemo-mechanical cycle of motor proteins?


Key Words: energy transduction • kinesin • mechano-chemical coupling • molecular mechanism • myosin

DIRECTED MOVEMENT IS one of the signs of life. It is expressed in a variety of ways in vivo. Macroscopic movement is fundamentally produced by the collective action of molecular motors; these use chemical energy to execute mechanical work. Understanding biomotor physicochemical mechanisms is important since it may contribute to curing of people suffering specific kinds of myopathies and neurological disorders. How do these molecular machines produce force, movement, and work? We concentrate on the proteins directly involved with contraction of the skeletal muscle—the most studied natural phenomenon in the field of mechano-chemistry.

Fibrous skeletal muscle cells contain numerous identical structural units, called sarcomeres. Their major structural characteristic is the interdigitation of thick (composed mostly of myosin-II) and thin (composed mostly of actin) filament arrays. It was established that a complex of only these two proteins, actomyosin, is sufficient to produce force and motility while hydrolyzing ATP (1) . The myosin filament is characterized by the ordered repetition of small globular bulges, or "heads" (~130 kDa), which hydrolyze ATP and bind actin, thus forming chemical cross-bridges, which enable force production (2 , 3) .

THE FRAMEWORK OF THE CTI MODEL

Several hypotheses had been put forward to elucidate the chemo-mechanical coupling within actomyosin. Myosin-centered hypotheses currently enjoy great interest among muscle scientists. The swinging lever-arm hypothesis is a popular myosin-centered hypothesis (1) . According to this hypothesis, ATP binding dissociates the myosin head from actin. A hydrolysis-coupled rotation of a lever-arm within myosin then occurs while myosin is free. A force is produced by reversal of the rotation after the myosin head rebinds to actin. The swinging lever-arm hypothesis does not elaborate on the specific nature of the conformational rearrangement that directly drives the lever-arm swing. It is assumed that myosin’s structural distortions throughout the biochemical cycle consist only of backbone bending caused by the twisting of {varphi} and {psi} peptide dihedrals. To the best of our knowledge CTI, a rotation of ~180° around the peptide bond dihedral {omega}, has not been addressed (Fig. 1 ). Among other uses, cis/trans isomerization is used for energy transduction. An example is phototransduction, which is mediated by isomerization of a carbon-carbon bond. This process is crucial for vision. Of interest is whether cis/trans isomerization is also used for the chemical-to-mechanical energy transduction responsible for directed movement.



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  		Figure 1. Nonproline and proline peptide fragments in trans and cis conformations.

OVERVIEW OF THE CTI MODEL

What is the specific conformational transition that propels the lever-arm swing? CTI is suggested here to be the driving mechanism within myosin and other motor proteins with a similar biochemical cycle. The CTI model resides within the framework of the swinging lever-arm hypothesis. It is assumed that a CTI of a specific peptide bond immediately follows ATP hydrolysis. Energy is internally transferred from the nucleotide binding pocket to the isomerizing peptide bond located within a CTI pocket. The route of energy transfer between the two pockets is unknown, so is the isomerization mechanism (4) . These are expected to be much clearer once the isomerizing bond is detected experimentally. The peptide bond first toggles from a trans conformation to a cis conformation. The tertiary structure may then be "locked in" if the energy barrier to the global energy minimum is relatively high. It is assumed that in skeletal myosin isoform the cis conformer is not further stabilized to a significant degree by the protein’s tertiary structure, the nucleotide, or its hydrolysis products. A spontaneous reverse interconversion slowly follows. For the peptide bond to isomerize, most of the free energy spread over the enzyme should focus into this specific peptide bond. Strong actin binding accelerates the reverse CTI, which then inflicts stress on actin. The cis conformer specifically and the whole myosin conformer in general are proposed to store the potential energy that later may be used to drive the actin slide (Fig. 2 ).



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  		Figure 2. Schematic representation of myosin structure during the proposed chemo-mechanical cycle that incorporates a cis/trans isomerization stage. NBP, nucleotide binding pocket; CTIP, CTI pocket.

We suggest that the motor element assumed to be common to myosin and kinesin is composed of a simple di- or a very short oligo-peptide segment. CTI probably occurs in an imide bond since its interconversion rate from cis to trans (~10–2 s–1) resembles myosin’s basal ATPase rate. Amide bond interconverts from cis to trans much faster, conversion rate being ~2 orders of magnitude higher, in case it is not stabilized. Amide cis conformer stabilization may be provided by the bound hydrolysis products or the surrounding residues. The search for the isomerizing bond should be guided by such considerations as evolutionary conservation, presence in different motor classes, proximity to the root of the lever-arm or nucleotide binding pocket, and neighboring residues that might facilitate isomerization. Referring to the amino acid sequence of Dictyostelium discoidium myosin, a protein homologous to the skeletal muscle myosin-II, we point to the following peptide bonds for further research: N219-P220, Q521-P522, N649-P650, and the amide bonds within the three conserved peptide segments that comprise the nucleotide binding pocket.

CIS/TRANS ISOMERIZATION AND ENERGY TRANSDUCTION

The significance of CTI for protein and cell function has been acknowledged only during the last three decades (5 , 6) . Several peptidyl-prolyl cis/trans isomerases and one secondary amide peptide bond isomerase were discovered (7 , 8) . CTI is suggested to have a major role in the biochemical cycle of several enzymes (9 , 10) , where substrate binding and product release are coordinated with isomerization. A concise account on the nature of the peptide bond isomers and their interconversion follows. Peptide bond CTI may occur in either an imide bond or an amide bond. Electronic delocalization stabilizes the peptide bond, i.e., lends it a moderate character between a single and a double bond (11) . This feature is expressed by the planarity of the peptide bond and in an energetic barrier (Ea) of ~55–100 kJ/mol against a rotation around the bond axis (6 , 12 13 14) . Ea depends on the specific amino acids combined in the peptide bond, conformation of its transition state, and the surroundings of the peptide bond. Relative to the trans conformation, the cis conformation may be destabilized by ~2–16 kJ/mol (13) . The peptidyl-prolyl cis conformer is relatively stable, destabilized only by ~2–4 kJ/mol relative to the trans conformation (4 , 8) . Destabilization of the cis conformation is demonstrated in oligopeptides and unstructured polypeptides. Because of the structural restrictions in folded proteins, often one conformation, either cis or trans is favored. In the native state thermal agitations may slowly drive polypeptide backbone interconversion. Spontaneous isomerization rate is the sum of the rate of the forward and the reverse process. There is no net production of cis (or trans) isomer in the equilibrium. The trans to cis spontaneous conversion rate is rather slow. The reverse conversion rate, cis to trans, is faster by ~3 orders of magnitude, amounting to ~10–3–101 s–1 at room temperature and pH 7 (13 , 15) . The rate constant of peptidyl-prolyl imide bond isomerization, cis to trans, is of the order of ~10–2 s–1. The interconversion rate may be accelerated dramatically by ~103–106 by introducing special protein surroundings for the isomerizing peptide bond, such as those provided by peptidyl-prolyl cis/trans isomerases (4 , 7) .

CORRELATIONS BETWEEN MYOSIN FUNCTION AND CTI

Kinetics
CTI is assumed to constitute the biochemical bottleneck of the myosin basal ATPase cycle. The rabbit fast skeletal myosin-II basal ATPase rate in vitro at room temperature is ~5·10–2 s–1 (16) , well within the range of spontaneous CTI in proteins. Actin binding in these conditions accelerates myosin ATPase by ~2 orders of magnitude to 10–20 s–1. The need for release of steric stress that develops due to strong actin binding is proposed to drive the cis conformer to isomerize back to the energetically low trans conformation: actin binding quenches myosin’s energized state. One may speculate that the relatively high Ea characterizing CTI may contribute significantly to the marked temperature dependence of the rate of ADP release (17) . The shape of the CTI pocket may heavily influence the spontaneous interconversion rate.

Energy barrier and energy supply
ATP hydrolysis under physiological conditions provides ~50–60 kJ/mol of free energy (18) . The energy barrier against CTI may be up to ~55–100 kJ/mol. These ranges overlap a little. Thus, energy supplied by ATP hydrolysis may be sufficient for overcoming the rotational barrier. Lowering Ea by ~50 kJ/mol may accelerate CTI rate by several orders of magnitude. The actual barrier height for CTI within a protein is highly dependent on the specific conditions within the CTI pocket, as evident in peptidyl-prolyl cis/trans isomerases that catalyze CTI without investing ATP energy.

Exerted force
Cis/trans isomerization is not just a switching mechanism but also a force generator. Advantage of this fact is taken in artificial motors (19 , 20) and in specific biomolecules, e.g., retinal isomerization within the restraining opsin protein. Retinal exerts force to distort the confining tertiary structure of the opsin (5) . The isomerizing peptide bond may be perceived as a nonlinear torsion spring. Rotation around the peptide bond produces a mechanical moment; from its magnitude a theoretical limit for force production by actomyosin may be derived. The strain energy added to the peptide bond due to an angle change ({theta}) in {omega} is proportional to Ea·sin2({theta}) (11) . Assuming the strain energy is not dependent on rotation rate, derivation by {theta} gives the expression for the mechanical moment, i.e., Ea·sin(2{theta}). The magnitude of the maximal moment produced is calculated to be ~Ea. This value translates to ~100 pN·nm per one motor protein for an Ea of ~60 kJ/mol. The average moment per stroke amounts to ~64 pN·nm. This value is obtained by integrating the expression for the moment by d{theta} and dividing by the integration interval, i.e., {pi}/2 for a rotation from {omega} = +90° to {omega} = +180°. Using this static model, one may assess the forces at the motor protein’s interface with its track produced by a pure moment acting within the motor core. Dividing the calculated average moment, i.e., ~64 pN·nm, by the length of myosin subfragment-1, i.e., ~16.5 nm (21) , gives an average reaction force of ~3.9 pN. Optical tweezing studies suggest that the force produced by a single head of myosin-II and an F-actin complex is in the range of 1–7 pN with an average value of 3.4 pN (22) . Calculations based on values derived from experiments conducted on muscle fibers contracting isometrically predict the average force per myosin head would be ~4 pN (23) . The measurement-derived values are similar to the theoretically derived value. The exerted force should get higher as the distance between the anchoring points, i.e., motor-track interface and motor-fix point (myosin thick filament), shortens; hence, significantly higher forces exerted by kinesin of ~6 pN may be explained.

Lever-arm swing
Rotation is the most straightforward mechanism for magnifying minute translations to large ones. CTI naturally produces a hinged movement. A recent Protein Data Bank survey of the local structural changes caused by peptidyl-prolyl CTI in proteins has suggested there is a high probability for a protein to undergo a considerable backbone expansion or contraction (C{alpha}/C{alpha} distances) during CTI (12) . The study further indicated that lever-arm amplification of isomerization mediated structural changes is possible. Stress relaxation around relatively free joints may abate the induced deflection. Rotation around {varphi} and {psi} peptide dihedrals requires overcoming a potential barrier of only 0.8–6.3 kJ/mol (24) . Peptidyl-prolyl cis conformer has a marked preference for turns and bends in the native state of many polypeptide chains. A conformational transition from such a bend or a turn to an extended structure containing a trans conformation is suggested to cause the lever-arm swing within myosin. Several experimental studies support a hinged-movement model for myosin (1) . Theoretically, the swinging lever-arm hypothesis postulates a lever-arm rotation of ~70° to allow for a myosin step relative to actin in the range of 5 to 16 nm. Thus, CTI induced rotation may explain experimental findings and theoretical predictions.

Motor directionality
Almost all known members of the myosin and kinesin families are unidirectional (25 , 26) , i.e., they stride along their track in one direction only. Most are plus-end directed; a few exceptions are myosin-VI and Ncd, a kinesin-like motor protein. A specific point mutation in Ncd abolishes directionality. KIF1A is another kinesin-like motor protein. It is inherently bidirectional. Phosphorylation might render some bidirectional biomotors, presumably dyneins, unidirectional (25) . What are the key structural factors determining directionality? Where do the direction-determining regions reside? According to the CTI model, motor directionality is determined by the direction of the peptide bond rotation due to its isomerization (trans to cis), which itself is dictated by the geometry of the CTI pocket and the relative orientation of the isomerizing peptide bond. In the reverse isomerization, pockets of plus end-directed motors induce rotation in one direction, say from {omega} = +90° to {omega} = +180° (i.e., generating positive moment); pockets of minus end-directed motors induce a relative counter-rotation, from {omega} = –90° {omega} = –180° (i.e., generating negative moment). The sign of the moment determines the direction of the reaction forces and hence motor directionality (Fig. 3 and Fig. 4 ). The aforementioned mutation in Ncd probably sabotaged the pocket’s geometry in such a way that neither rotation direction is preferred. KIF1A pocket is predicted to be neutral, i.e., the rotational barrier height is essentially the same for both rotation directions. The mentioned phosphorylation may induce an asymmetric CTI pocket; the barriers’ heights are expected to differ significantly after phosphorylation. The direction of a bond’s rotation due to cis/trans isomerization can be controlled. In fact, there is an artificial light-driven monodirectional molecular rotor (27) .



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  		Figure 3. The height of the rotational energy barrier vs. the torsional angle {omega} of a peptide bond. The direction of the rotation (cis to trans) is dependent on the relative height of EPOS and ENEG.



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  		Figure 4. The motors’ head conformations during the chemo-mechanical cycle. Isomerizing peptide bond in the middle. The root of the lever-arm follows the bond’s conformation. Lever-arm rotation of the two types of motors is through opposite arcs.

Chemo-mechanical decoupling
The coupling between ATP hydrolysis and actomyosin force production was suggested to be interrupted by various interventions, such as by mutating myosin or using different nucleotide analogs (28 , 29) . Energy supplying molecules may then be hydrolyzed at a slightly altered rate relative to normal conditions, but filament sliding may be severely retarded. The modifications may influence one or several critical physicochemical stages in the actomyosin cycle, e.g., the formation of an optimal configuration of the CTI pocket. Distortion of the CTI pocket may abolish CTI occurrence or its regulation.

SUGGESTED EXPERIMENTAL TESTS

So far, many myosin and kinesin structures have been solved with high resolution. Indirect indications for a lever-arm swing were provided by various crystallographic, microscopic, and spectroscopic studies (1 , 30) but it cannot be determined without a doubt whether the observed conformations are force generating or force bearing (see ref 31 ). Comparing the alleged pre- and post-powerstroke crystal conformations and searching for a cis conformer in them are probably not adequate for detecting CTI. There is no general agreement regarding what these structures represent and to what extent they are "pre" or "post." It is not yet resolved whether the initiation of the force-generating conformational transition precedes phosphate ion release, proceeds along with it, occurs between phosphate release and ADP release, or succeeds products release altogether. Further, the coupling between the chemical cycle and the mechanical cycle in myosin may not be tight (32) . The reverse CTI may well be loosely coupled to the release of hydrolysis products. One should not expect to see the induced cis conformation in available crystallographic structures because 1) these crystals either do not contain ATP, contain only poorly or nonhydrolyzable ATP analogs, or are in a nonhydrolyzing conformation (33) . The CTI model maintains that only after ATP hydrolysis does the isomerization take place. 2) The energized cis conformation is a transitory state, lasting only about a minute in the absence of actin. This short-lived state would not endure the process leading to crystallization. Therefore, one cannot use available X-ray structures of motor proteins to test the CTI model.

Site-specific incorporation of unnatural amino acids allows for the introduction of minute modifications within a protein‘s backbone or side chains (34) . These modifications may be used for specific labeling in spectroscopic studies (e.g., for averting the overlap inflicting NMR spectra) and for influencing the rotational stability of an interesting peptide bond. NMR mapping of sequential 1H-1H distances along the myosin backbone before and right after ATP hydrolysis may point to the hypothesized cis conformer. If the motor is driven by the isomerization of a specific peptidyl-prolyl imide bond, locking that bond in a specific conformation should paralyze the motor. One can lock the isomerizing imide bond by introducing various proline surrogates such as 2,4-methanoproline and 5,5-dimethylproline (35 , 36) or isosteric backbone modifications that significantly heighten the peptide bond’s rotational barrier, e.g., thioxo-de-oxo bisubstitution (37) . Subsequent functional alterations may be assessed by motility and ATPase assays.

The experiments suggested above are not decisive, but they do provide plausible leads for further investigation. How is it possible to distinguish if an eliminated synchronization of the motor protein cycle is due to the locked conformation or to an unwanted rearrangement induced in the surrounding tertiary structure by the amino acid surrogates? This fundamental question indicates the need of close monitoring and use of controls. We suggest using unnatural substitutes of proline similar stereochemically to proline. If the modification is minute (dihedrals, absolute dimensions, electrostatics, etc.), structural changes in the vicinity of the locked bond are expected to be insignificant. The tertiary structure of the modified protein should be analyzed (spectroscopy, microcalorimetry, etc.) for detecting significant unwanted rearrangements. As a control experiment, one may replace the native bond with a designed bond with a similar stereochemical structure, but this time the substituting bond would be relatively free to rotate (single unconjugated). Inserting minute modifications using unnatural amino acids should also help to identify and map other sensitive sites within the motor that are critical for its optimal function.

State-of-the-art biosynthetic techniques allow for the expression of an unnatural recombinant protein either in vitro or in vivo. For several years now, such an in vivo expression has been practiced using prokaryotic systems (34) . Recently, in vivo expression of unnatural proteins was demonstrated in the yeast Saccharomyces cerevisiae (38) , thus providing a gateway to the successful expression of eukaryotic proteins that otherwise would not correctly fold or be post-translationally modified. Unfortunately, myosin expression is currently restricted to eukaryotic hosts. This precludes production of large quantities of a modified protein in the near future. Thus, a prokaryotic in vivo expression system is preferable for producing adequate protein quantities using a relatively straightforward approach. Kinesin is an appropriate alternative for myosin since it could be expressed successfully using bacterial systems. The search for a transient cis conformation may be further facilitated in kinesin because it is a relatively small protein: only ~340 amino-terminal residues are sufficient for motility (39) .

Finally, it would be interesting to investigate the specific direct interactions, if any, of various cis/trans isomerases with unfolded and folded motor proteins in vitro. One might find that motor protein function is preserved for a longer duration and correct folding is assisted.

CONCLUSIONS

Elucidation of the physicochemical mechanism underlying motor protein function may allow controlling and redesigning these molecular machines for the benefit of medicine and technology. A novel and unifying model has been proposed, stating that the lever-arm swing assumed to occur within motor proteins during their functional cycle is driven by CTI. The following suggestions have been discussed: 1) ATP binding and hydrolysis provides energy for CTI in the myosin’s head; 2) a reverse CTI is the long postulated force generating conformational transition; 3) the reverse isomerization constitutes the biochemical bottleneck of the myosin’s basal ATPase cycle; and 4) actin binding to myosin accelerates the reverse CTI. At this stage the identity of the isomerizing bond, its location, and the isomerization’s reaction mechanism are unknown. A peptidyl-prolyl imide bond located at the catalytic domain is a promising candidate. Fundamental correlations between myosin’s function and peptide bond CTI have been discussed. Physical aspects covered are time, energy, force, lever-arm swing, motor directionality, and the functional mechanism. It is evident that the basic features of the CTI model are in accord with evidence available at present from studies of motor protein function. Peptide bond CTI has already been suggested to be a critical stage in the biochemical cycle of several enzymes. Given the difficulty of studying CTI, creative use of experimental techniques is required. Nonstandard site-directed mutagenesis may prove useful in the search for the assumed transient cis conformer. Preliminary tests of this model may be facilitated in kinesin. The model hereby suggested has been rendered falsifiable and provides "a path of opportunity" for experimental verification and challenge.

ACKNOWLEDGMENTS

The article is dedicated to my beloved parents—Marie (née Hakimzadeh) and Moshe Tchaicheeyan, the founder of TCHAICHEEYAN-Israel First College for Complementary Medicine. It is a pleasure to thank S. Har’el (Hebrew Univ., Jerusalem); J. A. Kennedy, A. Tzadok, and Dr. G. Ishai (Technion, Haifa); Drs. A. Oplatka and M. Elbaum (Weizmann Institute, Rehovot); and Dr. J. Abravanel (Paris, France) for stimulating discussions, criticism of early versions of the manuscript, and encouragement. I am particularly grateful to Dr. G. Ishai; without his help this endeavor could not have been undertaken. I thank Dr. A. Landesberg (Technion, Haifa) for introducing me to the muscle field. Thanks are due also to the Abravanel, Turtz, Potashnik, and Perlman families, who lit up the path.

Received for publication September 28, 2003. Accepted for publication January 26, 2004.

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