To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

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Conventional kinesin and Eg5 are essential nanoscale motor proteins. Single-molecule and presteady-state kinetic experiments indicate that both motors use similar strategies to generate movement along microtubules, despite having distinctly different in vivo functions. Single molecules of kinesin, a long-distance cargo transporter, are highly processive, binding the microtubule and taking 100 or more sequential steps at velocities of up to 700 nm/s before dissociating, whereas Eg5, a motor active in mitotic spindle assembly, is also processive, but takes fewer steps at a slower rate. By dissecting the structural, biochemical and mechanical features of these proteins, we hope to learn how kinesin and Eg5 are optimized for their specific biological tasks, while gaining insight into how biochemical energy is converted into mechanical work.

Introduction

Microtubule-based kinesin superfamily motors are involved in diverse cellular processes including intracellular transport, mitosis and meiosis, regulation of microtubule dynamics, and signal transduction [1]. Here we consider conventional kinesin, (‘kinesin’, the Kinesin-1 subfamily) and Eg5 (the Kinesin-5 subfamily), both of which have conserved N-terminal catalytic motor domains and walk processively toward the plus-end of microtubules, hydrolyzing one ATP per 8-nm step ([2, 3, 4••], reviewed in [5•, 6, 7, 8•, 9]).

In both, the motor domain is followed by a 12–15 amino acid residue neck linker leading to the coiled-coil stalk. However, there are important structural differences (Figure 1). Kinesin is a homodimer, composed of two heavy chains, with two light chains associating with the C-terminal cargo-binding domain. By contrast, Eg5 is a homotetramer: two polypeptides first dimerize to form a parallel coiled-coil, and then two dimers form an anti-parallel coiled-coil tetramer containing four motor domains. As a tetramer, Eg5 can crosslink two adjacent microtubules such that each dimeric motor unit interacts with a single protofilament on each microtubule [10•, 11••].

Kinesin was discovered in 1985 [12] and new single molecule assays [13, 14] and presteady-state kinetic experiments [15] soon followed. Since then, thousands of experiments have been performed, allowing a consensus model to emerge (Figure 2). By contrast, mechanochemical data for Eg5 are just beginning to appear. Here, we review the current model for kinesin processivity, summarizing the evidence for a strain-gated hand-over-hand mechanism, and then examine what we currently know about the kinetics and mechanics of Eg5 (Figure 2, Table 1), highlighting recent advances and unresolved questions for each.

Section snippets

A consensus mechanochemical cycle for dimeric kinesin

In solution, each of kinesin's motor heads contains a tightly bound ADP, which is released upon collision with the microtubule. This rapid release is biphasic: the first ADP is released at >200 s−1, while the release of the second ADP is nucleotide-dependent, with ATP-stimulated release at >100 s−1 (Table 1, [16, 17, 18, 19]). When interpreted in the context of the Rice et al. neck linker model [20, 21, 22], these data lead to the following pathway (Figure 2, steps K1–K4). The first motor head

Kinesin processivity: unresolved questions

Although the model presented in Figure 2 is representative of published results, there remain points of disagreement and steps that require stronger experimental support. Between the conformational changes that drive processive stepping, the motor pauses in a ‘waiting’ state, the nature of which is largely unknown but which is likely to depend on the ATP concentration and applied load. One point of controversy is the position of the tethered head with respect to the bound head and microtubule

An emerging model of Eg5 mechanochemistry

To determine the mechanical and kinetic requirements for Eg5-promoted spindle assembly [47, 48, 49], a stable Eg5 dimer was developed [11••]. Eg5-513 promoted robust plus-end-directed microtubule gliding at a rate similar to that of native tetramers [11••, 50]. Single Eg5 dimers were found to step processively in optical trapping experiments, taking approximately eight steps on average at speeds of up to 100 nm/s at saturating ATP levels and zero applied load [4••]. Moreover, Eg5 dimers

Eg5 processivity: the challenge awaits

Eg5 dimers are clearly processive, but take far fewer steps than kinesin under similar conditions. A key area of future research will be determining what limits the run length of Eg5. Two possibilities seem likely: that the microtubule-binding affinity of each Eg5 motor head is weak enough in all nucleotide states to promote frequent detachment from the microtubule, or, alternatively, that strong binding states exist, but that poor head–head communication prevents the tight alternation of the

Conclusions

Our understanding of kinesin superfamily members is advancing rapidly, in part because of technological improvements, but also because of the widespread interest in these molecular motors. They are present in every cell of every eukaryotic organism, and are intimately involved in human health and development. Comparisons of conventional kinesin and Eg5 show that in spite of their structural and mechanistic similarities, each has optimized its chemical and mechanical cycle differently for

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Nick Guydosh, Troy Krzysiak, Andreas Hoenger, and Steve Rosenfeld for helpful discussions and careful reading of the manuscript, and Polly Fordyce for assistance with Figure 1. M.T.V. was supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. S.P.G. was supported by grant GM54141 from NIGMS and Career Development Award K02-AR47841 from NIAMS, National Institutes of Health.

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