Neck Linker Docking Research Articles

Monte Carlo Analysis of Neck Linker Extension in Kinesin Molecular Motors

Kinesin stepping is thought to involve both concerted conformational changes and diffusive movement, but the relative roles played by these two processes are not clear. The neck linker docking model is widely accepted in the field, but the remainder of the step – diffusion of the tethered head to the next binding site – is often assumed to occur rapidly with little mechanical resistance. Here, we investigate the effect of tethering by the neck linker on the diffusive movement of the kinesin head, and focus on the predicted behavior of motors with naturally or artificially extended neck linker domains. The kinesin chemomechanical cycle was modeled using a discrete-state Markov chain to describe chemical transitions. Brownian dynamics were used to model the tethered diffusion of the free head, incorporating resistive forces from the neck linker and a position-dependent microtubule binding rate. The Brownian dynamics and chemomechanical cycle were coupled to model processive runs consisting of many 8 nm steps. Three mechanical models of the neck linker were investigated: Constant Stiffness (a simple spring), Increasing Stiffness (analogous to a Worm-Like Chain), and Reflecting (negligible stiffness up to a limiting contour length). Motor velocities and run lengths from simulated paths were compared to experimental results from Kinesin-1 and a mutant containing an extended neck linker domain. When tethered by an increasingly stiff spring, the head is predicted to spend an unrealistically short amount of time within the binding zone, and extending the neck is predicted to increase both the velocity and processivity, contrary to experiments. These results suggest that the Worm-Like Chain is not an adequate model for the flexible neck linker domain. The model can be reconciled with experimental data if the neck linker is either much more compliant or much stiffer than generally assumed, or if weak kinesin-microtubule interactions stabilize the diffusing head near its binding site.

Kinesins at a glance.

Kinesin was discovered in 1985 – 25 years ago – based on its motility in cytoplasm extruded from the giant axon of the squid ([Allen et al., 1982][1]; [Brady et al., 1982][2]; [Vale et al., 1985][3]). The purified protein, now known as kinesin-1, consisted of two heavy chains with motor activity

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.

The Conserved L5 Loop Establishes the Pre-Powerstroke Conformation of the Kinesin-5 Motor, Eg5

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.

1P182 1B1325 キネシン1が選択的に前方にステップする仕組み(分子モーター,口頭発表,第48回日本生物物理学会年会)

1P182 1B1325 キネシン1が選択的に前方にステップする仕組み(分子モーター,口頭発表,第48回日本生物物理学会年会)

The Neck Linker Docking is Not Required for Kinesin-1 to Take a Step Forward

Kinesin-1 moves processively along microtubules by alternatively moving two motor domains, but the mechanism of the preferential forward stepping is still controversial. The “neck linker-docking model” proposes that the neck linker of the microtubule-bound head propel the tethered head forward. We proposed an alternative “biased-capturing model” that the tethered head freely diffuses and is captured preferentially at the forward-binding site. The latter model predicts that the neck linker of the tethered head, not of the microtubule-bound head, is essential for stepping, but it is difficult to distinguish these effects using “symmetric kinesin dimer”.

Neck-Linker Length is a Critical Determinant of Kinesin Processivity

The kinesin neck-linker domain is a key mechanical element underlying processive kinesin motility. Not only is neck-linker docking thought to be the dominant conformational change in the kinesin hydrolysis cycle, chemomechanical communication between the two head domains must necessarily be transmitted through the two neck-linker domains and their shared coil-coil. Hence, the length of the neck-linker is expected to have a strong influence on kinesin run length, a quantitative measure of processivity. Across different kinesin families, motors with longer neck-linker domains, such as Kinesin-2 are generally less processive than Kinesin-1, which has the shortest neck-linker domain among N-terminal kinesins. However, there is disagreement in the literature as to whether artificially extending the Kinesin-1 neck-linker alters the motor run length. Using single molecule TIRF analysis to visualize GFP-tagged motors in 80 mM PIPES buffer, we find that lengthening the Kinesin-1 neck-linker by three amino acids results in a five-fold reduction in run length. Consistent with this, when the Kinesin-2 neck linker was matched to the effective length of Kinesin-1 by deleting three residues and substituting an alanine for a proline, the Kinesin-2 run length nearly matched that of Kinesin-1. These results demonstrate that run length scales with neck linker length for both Kinesin-1 and Kinesin-2 and is sufficient to account for differences in processivity. In addition, we find that adding positive charge to neck linker inserts enhances processivity, providing a possible explanation for the lack of dependence of run length on neck-linker length observed by others. Our data is consistent with the hypothesis that increasing neck linker compliance reduces processivity by disrupting front head gating and potentially provides a unifying principle across kinesin families - longer neck-linkers lead to less processive motors.

Coupling of Kinesin-1 Neck Linker Docking to the Nucleotide Binding Site

Coupling of nucleotide binding to docking of the neck linker of kinsin-1 is important for generation of directional motility. One approach towards determining the magnitude of this coupling is to use isotopic exchange reactions to evaluate the free energy differences between states. Kinesin-1 monomer head domains catalyze the slow MT-dependent synthesis of bound ATP from bound ADP and free Pi ( MT•E•ADP + Pi → MT•E•ATP + HOH) that results in oxygen isotopic exchange of 18O/16O between water and Pi. The tethered head domain of a kinesin dimer bound to MTs, however, catalyzes ATP synthesis at a 20-fold faster rate (Proc.Natl.Acad.Sci.USA 102, 18228 (2005)). This more rapid rate of ATP synthesis with a dimer suggests that the tethered head can bind to the microtubule behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the microtubule and would facilitate its docking on synthesis of ATP.Isotopic exchange analysis of other constructs with alterations in the neck linker is in progress. One approach is to delete part of the neck linker and therefore prevent reversible docking. DKH335 has lost the C-terminal part of the neck linker that makes extensive contacts with the core. During net ATP hydrolysis, the full length head domain DKH346 resynthesizes ATP on average once in 40 turnovers. In contrast, DKH335 is reported here to hydrolyze ATP with no detectable ATP resynthesis (ATP resynthesis occurs only once in >500 turnovers). This is consistent with more rapid Pi release in the absence of a requirement for coupled neck linker undocking or with destabilization of bound ATP in the absence of neck linker docking.Supported by NSF grant MCB-0615549.

The ATPase Cycle of the Mitotic Motor CENP-E

We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding. These features endow CENP-E with a high duty cycle, a prerequisite for processivity. However, unlike kinesin 1, neck linker docking in CENP-E is slow, occurring at a rate closer to that for Eg5, a mitotic kinesin that takes only 5-10 steps per processive run. These results suggest that like kinesin 1, features outside of the catalytic domain of CENP-E may also play a role in regulating the processive behavior of this motor.

Hopping and stalling of processive molecular motors

When a two-headed molecular motor such as kinesin is attached to its track by just a single head in the presence of an applied load, thermally activated head detachment followed by rapid re-attachment at another binding site can cause the motor to ‘hop’ backwards. Such hopping, on its own, would produce a linear force–velocity relation. However, for kinesin, we must incorporate hopping into the motor's alternating-head scheme, where we expect it to be most important for the state prior to neck-linker docking. We show that hopping can account for the backward steps, run length and stalling of conventional kinesin. In particular, although hopping does not hydrolyse ATP, we find that the hopping rate obeys the same Michaelis–Menten relation as the ATP hydrolysis rate. Hopping can also account for the reduced processivity observed in kinesins with mutations in their tubulin-binding loop. Indeed, it may provide a general mechanism for the breakdown of perfect processivity in two-headed molecular motors.

Water in the Neck-Zipper Region of Kinesin

Neck linker (NL) is one of the most important mechanical elements of kinesin motors. The zipping up process of the neck-zipper (NZ) formed by NL and the related secondary structure elements is one of the major parts of kinesin's power stroke. All the weak interactions that are responsible for the formation of NZ are sensitive to or dependent on water. To investigate the role of water in the NZ region, a molecular dynamics (MD) model is set up with a crystal structure of kinesin 2KIN surrounded by a 10 Å water layer, and minimization is performed to determine the positions of hydrogen atoms and other atoms. It is revealed that water molecules can assist the docking process of NL by forming hydrogen bonds at those positions where direct hydrogen bonding between the two sides of NZ is hindered and then acts as a constructive component of NZ at the docked state of NL. This result may improve the understanding of the mechanism for the docking of NL of kinesin wherein the function of water has not been comprehended sufficiently.

Processive Motility of Heterodimeric Kinesin That Has Defect in the Neck Linker Docking

Kinesin-1 is a motor protein that moves processively along microtubules in a hand-over-hand manner. The neck linker, a short stretch that connects two motor heads, has been shown to undergo ATP-dependent conformational changes, although its role on the processive motility is still controversial. To address this question, we employed a kinesin mutant at switch I (R203K) that can normally bind ATP but is unable to hydrolyse ATP (Klumpp et al. JBC 2003). First we observed the neck linker structural state of monomeric R203K bound to the microtubule using single molecule FRET. We found that the neck linker remained undocked conformation even in the presence of saturating ATP, suggesting that ATP-binding is not sufficient to stabilize neck linker docked state. Next we constructed heterodimeric kinesin that is composed of a wild-type head and a mutant R203K head. As recently been shown by Thoresen and Gelles (Biochemistry 2008), this heterodimer showed slow processive movement along microtubules. Then we observed the conformational changes of this heterodimer using a single molecule FRET sensor as previously developed to distinguish one-head-bound and two-head-bound states (Mori et al. Nature 2007), and found that the heterodimer showed hand-over-hand movement. Unexpectedly, they spent most of the time in the two-head-bound state where wild-type head is in the front and the mutant head is in the rear, indicating that the displacement of rear wild-type head to the forward binding site is not the rate-limiting. These results suggest that the neck linker docking in the microtubule-bound head is not essential for the tethered head to translate and bind to the forward tubulin-binding site and rather is required for promoting ATP-hydrolysis and subsequent detachment of the trailing head from microtubule.

The ATPase Cycle of the Mitotic Motor CENP-E

Several members of the kinesin superfamily are highly processive molecular motors, capable of taking multiple steps on their track without dissociating. For the processive transport motor kinesin I, processivity results from features of this motor's enzymology, which enhance its ability to stay attached to the microtubule through multiple ATPase cycles. These include a high duty ratio, rapid rate of ATP binding, rapid rate of forward stepping, and strain-dependent gating of nucleotide binding. We had recently shown that CENP-E, a mitotic kinesin that localizes chromosomes to the midzone during metaphase, is highly processive, with run lengths similar to those for kinesin I. In this study, we have examined the CENP-E ATPase cycle to see if its enzymology explains its processive behavior. We find that some features of the CENP-E enzymatic cycle, including rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and strain-dependent gating of nucleotide binding, are indeed very similar to those for kinesin I. However, the rate of neck linker docking for CENP-E is nearly 20-fold slower, and this would predict run lengths 50-fold shorter than what we have observed. This implies that, like the mitotic motor Eg5, CENP-E processivity is shaped not only by its enzymology, but also by other, non-enzymatic features of this molecular motor as well.

The relevance of neck linker docking in the motility of kinesin

Conventional kinesin is a motor protein, which is able to walk along a microtubule processively. The exact mechanism of the stepping motion and force generation of kinesin is still far from clear. In this paper we argue that neck linker docking is a crucial element of this mechanism, without which the experimentally observed dwell times of the steps could not be explained under a wide range of loading forces. We also show that the experimental data impose very strict constraints on the lengths of both the neck linker and its docking section, which are compatible with the known structure of kinesin.

1P-153 ネックリンカーが正常にドッキングできないヘテロニ量体キネシンの連続歩行運動(分子モーター(1),第46回日本生物物理学会年会)

1P-153 ネックリンカーが正常にドッキングできないヘテロニ量体キネシンの連続歩行運動(分子モーター(1),第46回日本生物物理学会年会)

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.

Kinesin: a molecular motor with a spring in its step.

A key step in the processive motion of two-headed kinesin along a microtubule is the 'docking' of the neck linker that joins each kinesin head to the motor's dimerized coiled-coil neck. This process is similar to the folding of a protein beta-hairpin, which starts in a highly mobile unfolded state that has significant entropic elasticity and finishes in a more rigid folded state. We therefore suggest that neck-linker docking is mechanically equivalent to the thermally activated shortening of a spring that has been stretched by an applied load. This critical tension-dependent step utilizes Brownian motion and it immediately follows the binding of ATP, the hydrolysis of which provides the free energy that drives the kinesin cycle. A simple three-state model incorporating neck-linker docking can account quantitatively for both the kinesin force-velocity relation and the unusual tension-dependence of its Michaelis constant. However, we find that the observed randomness of the kinesin motor requires a more detailed four-state model. Monte Carlo simulations of single-molecule stepping with this model illustrate the possibility of sub-8 nm steps, the size of which is predicted to vary linearly with the applied load.