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Biology Articles » Biophysics » Medical Biophysics » The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease » Applications of the Unified Theory to Other (Processive) Molecular Motors Crucial toCell Life

Applications of the Unified Theory to Other (Processive) Molecular Motors Crucial toCell Life
- The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

The cytoplasm of cells is crowded with cargoes that are being moved processively along tracks of microtubules or actin by various molecular motors of the kinesin, myosin and dynein family. Efficient translocation of vesicles and membranous organelles such as mitochondria and endoplastic reticulum over micrometer distances is an activity crucial to cell life. The best-characterized unconventional myosin motor stepping processively on actin filaments is myosin V, which is involved in several types of intracellular transport. Kinesin constitutes a very large family of motor proteins that transports cargo processively on microtubules at speeds as high as ~1 μm/s, and there are as many as ~50 different kinesins in humans [61]. The best-characterized so-called conventional kinesin is abundant in nerve cells. It is double-headed and has an N-terminal motor domain, a coiled coil stalk, and a C-terminal cargo-binding domain, and transports cargo processively towards the microtubule plus end, i.e., toward the cell membrane. A central issue in the field of molecular motors has been to understand how the chemical and mechanical steps are coupled and how processive motion occurs using the energy of ATP hydrolysis in such conventional kinesin and myosin V motors. Various models have been developed and different mechanisms proposed to explain processive motion by these molecular motors.

Due to space limitations, it is not possible to study the scores of each and every variant of kinesin, ncd and myosin V models for processive motility and compare and contrast them. However, the various alternative mechanisms can be classified, their overall features understood, and the unified theory applied to the mechanism or model of choice. It is well known that kinesin, ncd and myosin V heads are structurally equivalent. Hand-over-hand models of processive movement postulate equivalence of the two heads, i.e. not that the heads are structurally equivalent, which is well-known and understood as stated above, but that the heads are functionally equivalent, i.e., the same biochemical (especially ATPase) events occur in both heads except that these events alternate and lag in time. A subset of these models, called symmetric hand-over-hand models [62, 63] also include the key proposal of step equivalence, i.e. that all steps are generated in the same way. Thus in symmetric hand-over-hand models, the rotation is 180o in a clockwise or counterclockwise sense each time, and the stepping head passes the head attached to the track from the same side (left or right) each time. Thus, symmetric hand-over-hand models postulate head equivalence and step equivalence [62, 63]. However, experimental data from the Gelles group contradicted the above because the postulated 180o swiveling or rotation was not observed with each step [64]. To explain these observations, head equivalence was abandoned and an inchworm model was proposed [64] in which the heads were functionally inequivalent in that only one head was an active ATPase, and one head always led the other, but the mechanism retained step equivalence. Thus the inchworm model is head inequivalent but has step equivalence. Based on single molecule experiments, Block and colleagues postulated an asymmetric hand-over-hand model of kinesin movement [65] in which the two heads are functionally equivalent and both hydrolyze ATP alternately, but the steps are non-equivalent in that the stepping head passes the attached head on alternate sides and the stalk makes compensatory movements to suppress the 180o rotations. Hence the asymmetric hand-over-hand model has head equivalence but does not have step equivalence. However, the head equivalence postulated by the asymmetric handover- hand model is challenged by the observation that heterodimers with ATPase mutation in only one kinesin head move processively [66]; in other words, processive motion is possible without alternating catalysis. Further, there exist difficulties for the asymmetric hand-over-hand model in explaining the exact cause of limping by homodimeric kinesin [65] in which no structural asymmetry is apparent. Based on molecular systems biology approaches, Nath has proposed a rotation-twist energy storage mechanism for processive molecular motors such as kinesin, ncd and myosin V [11], which was subsequently developed in further detail [10]. In this mechanism, one head of the motor binds weakly to the α-tubulin subunit on the microtubule or to actin while the other head binds strongly to the β- tubulin subunit on the microtubule or to actin. To take the example of kinesin, only the head bound to the β-tubulin subunit on the microtubule is an active ATPase because the interactions of the α-tubulin with the bound kinesin head are not strong enough to release the MgADP bound in that head; hence no ATP hydrolysis occurs in the head bound to the α-tubulin subunit on the microtubule. Further, the two steps occur due to different causes; the first one occurs due to ATP binding and its subsequent hydrolysis (called “the chemical stroke” [10, 11]), while the cause of the second step is the torsional strain in the V-shaped molecule up to hinge-1 due to the ATP hydrolysis Coulombic energy released upon Pi release, though the step itself takes place after the trigger of ADP release from the head and the release of the torsional strain (called “the physical stroke” [10, 11]). In the physical and chemical strokes, the rear head passes the bound front head from different sides and the strokes have different trajectories; moreover, since the strokes originate from different causes, they differ in their kinetics, and thus the rotation-twist energy storage mechanism for processive molecular motors has head inequivalence and step inequivalence. The mechanism has no difficulty in explaining the observations in Ref. 66 because in the mechanism, only one head hydrolyzes an ATP molecule to move the doubleheaded molecule processively by ~8 nm, and further, the progressive shortening of the coiled coil in the truncated constructs [65] hamper the storage of elastic strain in the molecule which adversely affects the performance of the physical stroke and therefore increases the propensity of the molecule to limp. The mechanism is simpler and more elegant and is ideally expected to operate in vivo. The mechanism can readily be adapted to the case in vitro when both heads bind to β-tubulin subunits on the microtubule, the only difference being that ATP binding will now be required to unbind the second head also (i.e. the head that had bound earlier to the α-tubulin subunit on the microtubule), as discussed later, in which case the mechanism can be said to have head equivalence but no step equivalence. It should be noted that there is considerable experimental evidence for specific interaction of kinesin and ncd motor heads with both α- and β-tubulin [67-70]. Moreover, the weaker affinity of the specific binding site on α-tubulin may prevent its identification, or it is likely that both the heads may preferably bind to the higher-affinity specific sites on β-tubulin in vitro, and another positive point of the new mechanism is that it assigns a role to the second weaker but specific binding site identified by biochemical labeling and electron microscopy techniques [67-70] in motor function. The function of these weaker, specific motor-binding sites on the microtubule that have been revealed by experiment had hitherto been unknown. Finally, it should also be noted that the inchworm model was also defective because it did not solve the difficult problem of how the motor moved both heads by hydrolyzing a single ATP molecule. This is not a defect in the new mechanism.

Recent fluorescence labeling and single molecule studies from Selvin’s group have clearly invalidated the inchworm model of processive motion and shown that kinesin moves in a hand-overhand fashion [71]. Other models have also been proposed in which the two heads are 4 nm apart [72, 73], and Hackney’s classical experimental data also mean that there are two motor head binding sites with different affinities on the microtubule [74, 75]. These models are unable to explain the 16 nm steps seen in single molecule studies, which imply that the two heads of kinesin are 8 nm apart [71]. The limping seen in homodimeric kinesin has proved very difficult to explain, and Selvin and colleagues have recently stated that kinesin could indeed walk symmetrically and that the observed limping was due to the experimental conditions [76]. Unfortunately, their explanation contradicts the experimental data of the Gelles group [64]. Impressive evidence that kinesin moves by an asymmetric hand-over-hand model has also been produced recently [65, 77], and, in particular, Block and colleagues favor a gated front head model [77].

However, in addition to the question of limping by homodimeric kinesin constructs which has not been satisfactorily answered to date by the asymmetric hand-over-hand model of motility, there is another even more fundamental and central question that has not been asked till now, but needs to be asked now and sorely requires an unequivocal answer. This question is the following: Does kinesin move forward processively in steps of 16 nm head-to-head (i.e. 8-nm steps for movement of the center of mass of the double-headed molecule) against a load of ~7 pN, the maximum force against which kinesin molecules have been consistently recorded to step forward in various expertly-performed stateof- the-art single molecule experiments [72, 78-80], and not ~half the load of ~7 pN, i.e. ~3.5-4 pN, where it is certainly known to move forward processively with 8-nm (center of mass) steps [65]? Answering this question is crucial because in the optical trap experiments, for constant energy input from ATP, it is readily possible to satisfy the energy balance by reducing the force by a factor of ~2 and thereby obtain an increase in the step size by a factor of ~2. In a way, this question has already been answered by single molecule experiments on kinesin at an intermediate force range of 4.5-5.3 pN, where already, kinesin starts to step backwards [77]. Hence the all-important question arises: Why does kinesin step backwards at intermediate loads of 4-5 pN, well below the experimentally measured stall force of ~7 pN? In fact, it should have stepped forward processively even at a ~7 pN rearward load, in accordance with the single molecule experiments of several groups, including Block’s own group [72, 78-80]. Unless the step size (8 nm based on center of mass movement) in these in vitro experiments is larger than the actual step size in vivo, and therefore the energy of ATP is insufficient to supply the energy for processive forward stepping against the intermediate rearward load of even 4- 5 pN (let alone ~7 pN), and therefore the rearward load wins and pulls the kinesin molecule backwards, it is not possible to explain the observation of backward stepping at these intermediate loads, according to our analysis. In the absence of a demonstration of processive forward stepping with 16 nm head-to-head steps at the maximum load against which kinesin molecules are known to move forward processively [72, 78-80], the proposal of hand-over-hand models that the molecule always advances by 16 nm head-to-head steps (8 nm movement of the center of mass) is significantly weakened. I predict that this question will prove to be the Achilles heel for the asymmetric hand-overhand model. Ideally, what is needed is a molecular mechanism for forward processive motility that works up to the maximum force (~7 pN for kinesin) and not a mechanism that works only up to ~half the maximum force, and when the load is increased slightly beyond this value, the motor begins to move in the backward direction. The rotation-twist (RT) energy storage mechanism for processive molecular motors offers the further advantage that it explains forward processive motion under vertical loading that we expect in vivo due to the presence of the cargo at the C-terminal end of kinesin. It should also be pointed out that the asymmetric hand-over-hand model is not the only model that includes alternation between two different configurations during processive stepping; the RT mechanism also shares this attribute [10, 11]. Hence we believe that the rotation-twist (RT) energy storage mechanism for processive molecular motors should also be included among candidate mechanisms in the field of motility along with symmetric and asymmetric hand-over-hand and inchworm models.

The RT mechanism is readily able to explain the experimental facts [62-80] and also possesses several new and attractive features. Thus the data on processive movement of kinesin against the maximal load of ~7 pN [72, 78-80] is explained by the RT mechanism by postulating that in these experiments the two kinesin heads must have bound to α- and β-tubulin on the microtubule. In the experiments in which the two heads are bound to adjacent β-tubulin subunits on the microtubule, forward processive motion can readily occur at a load value up to approximately half the maximum load, with the larger step size of 16 nm (head-to-head), as observed [65]. Thus, forward processive motion is possible in both cases, but not against the high ~7 pN load in the case when both kinesin motor heads bind to β-tubulin subunits on the microtubule. Thus, from the point of view of the RT mechanism, kinesin can move with 4 nm center of mass (= 8 nm head-to-head) steps or with 8 nm center of mass (= 16 nm head-to-head) steps. The two cases depend on whether one head binds to β- tubulin and the other to the adjacent α-tubulin subunit of the microtubule track (former case) or whether both heads bind to adjacent β-tubulin subunits of the microtubule track (latter case). Since a motor head is bound weakly to α-tubulin (compared to the binding of the counterpart motor head bound to β-tubulin), it does not require MgATP binding to help its release from the microtubule, as the stored strain energy in the molecule after the chemical stroke and release of Pi is sufficient by itself to release such a (rear) head bound to α-tubulin [10, 11]. This is also consistent with the longstanding observation of the presence of two motor head binding sites with different affinities on the microtubule [74, 75]. On the other hand, if both motor heads bind to β-tubulin, then the stored strain energy is not sufficient to release the rear head bound to β-tubulin and requires help from another energy source, which we propose to be MgATP binding to the rear head in the RT mechanism. Applying the unified theory to the RT mechanism, we can state with full confidence that the first (rear) head is moved (past the bound front head) from one side by a clockwise rotation viewed from the C-terminal cargo end of conventional kinesin due to the ~9 kJ/mol MgATP binding energy (i.e. the surplus energy released over that required to unbind the head from the microtubule) plus the ~9 kJ/mol energy released upon the process of hydrolysis of the MgATP (that had just bound in that head) that occurs when the head is free from the microtubule track (and before Pi release, which requires binding of the head to the next binding site on the track). This is the “chemical step” [10, 11]. In the “physical step” [10, 11], the Coulombic repulsion energy between MgADP and Pi released upon Pi release into the medium (~18 kJ/mol according to the unified theory) causes an elastic (primarily torsional) strain that helps move the second head (the present rear head) past the bound front head from the other side (compared to that in the earlier “chemical step”) by a counterclockwise rotation viewed from the C-terminal cargo end either without requiring MgATP binding in the second (rear) head in the case when the head is bound to α-tubulin, or also requiring MgATP binding in the rear head to help release the head in the case when it is bound to β-tubulin. The limping of homodimeric kinesin [65] is readily explained by the RT mechanism because as the kinesin stalk is progressively shortened, the energy storage process in the molecule is progressively hampered and the torsional rigidity required to generate torque by the physical stroke about hinge-1 [10, 11] is progressively compromised.

In the RT mechanism, MgATP binding to the rear head releases binding energy which unbinds the rear head from the microtubule track and gives it a clockwise torque (seen from the C-terminal cargo end), and it is perfectly reasonable that when the head starts to slow down or even pauses, as the sharp input of energy due to the release of MgATP binding energy in the head that initiated the chemical step gradually loses its effect, bond cleavage of MgATP occurs in the head and the release of the requisite quantum of hydrolysis energy (as detailed in the unified theory) continues to drive the head to its next binding site on the microtubule and helps it complete the chemical step, i.e. MgATP binding and MgATP hydrolysis acting in sequence cause the chemical stroke. This is a superior proposal to the central tenet of the gated front head model in which MgATP binding to the front head causes the rear head to step forward [77]. The RT energy storage mechanism also differs from the gated rear head model in which Pi release from the rear head (but not ATP binding to the rear head or the subsequent ATP hydrolysis that is postulated to occur in the rear head while it is still bound to the microtubule) causes the rear head (now containing bound ADP) to step forward past the bound front head. In both gated front head and gated rear head models within the hand-over-hand mechanisms, internal strain in the kinesin molecule is only used to reduce the affinity (of the leading head for ATP in the gated front head model, or of the rear head for the microtubule in the gated rear head model). It is only in the RT mechanism that elastic (twisting) strain energy is stored in the molecule and used to cause the forward stepping of the rear head past the bound front head upon release of the stored strain energy, i.e., for performance of useful external work, which in our opinion is a great leap in our thinking and a progressive step forward for models of processive motility. In this sense, the RT energy storage mechanism and the symmetric or asymmetric hand-over-hand models belong to different classes of models of processive motility.

Finally the new molecular mechanism for processive motility is simpler, contains fewer biochemical steps, is aesthetically pleasing and elegant, and, especially significant in the context of processive motors, faster than previous models of processivity. The aspects discussed in this section are also applicable to unconventional myosins, and in fact, the mechanism has also been readily adapted to explain the processive motion of unconventional myosins such as myosin V on actin filaments [10, 11] and in the field of myosin motility, it may play an important progressive role. A key achievement of the RT energy storage mechanism in unconventional myosin motility is that it offers an increase of efficiency of intracellular transport by a factor of two over all previous mechanisms, a very large and significant improvement [11]. This is because, in all previous mechanisms, two ATP molecules are consumed for a forward movement of the center of mass of the double-headed myosin by 72 nm, while in the RT energy storage mechanism only a single ATP molecule is used to power the center of mass of the myosin forward by 72 nm. An identical rotation-twist energy storage molecular mechanism applies to the motility of the non-claret disjunctional (ncd) motor, with one extra turn in the ncd neck coiled coil (compared to kinesin) constituting the relaxed state of ncd, thus accounting for movement of the ncd motor towards the minus end of the microtubule. In conclusion, it can be stated that this section has clearly indicated a large number of useful applications of the unified theory to various processive molecular motors involved in intracellular motility, which is crucial to cell life.

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