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Kinesin's cover-neck bundle folds forward to generate force.

ABSTRACT: Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a beta-sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the N-terminal "dangling end," unlike the neck linker and nucleotide-binding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.

SUBMITTER: Khalil AS

PROVIDER: S-EPMC2592363 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Neck-linker docking coordinates the kinetics of kinesin's heads.

Project description:Conventional kinesin is a two-headed homodimeric motor protein, which is able to walk along microtubules processively by hydrolyzing ATP. Its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. Although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. Considering the two neck linkers as entropic springs that are allowed to dock to their head domains, and incorporating only the few most relevant kinetic and structural properties of the individual heads, we develop here the first, to our knowledge, detailed, thermodynamically consistent model of kinesin that can 1), explain the cooperation of the heads (including their gating mechanisms) during walking, and 2), reproduce much of the available experimental data (speed, dwell-time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck-linker length and composition, etc.). Besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion.

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Kinesin's front head is gated by the backward orientation of its neck linker.

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In vivo optical trapping indicates kinesin's stall force is reduced by dynein during intracellular transport.

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Kinesin's neck-linker determines its ability to navigate obstacles on the microtubule surface.

Project description:The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin's processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.

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Polymerisation force of a rigid filament bundle: diffusive interaction leads to sublinear force-number scaling.

Project description:Polymerising filaments generate force against an obstacle, as in, e.g., microtubule-kinetochore interactions in the eukaryotic cell. Earlier studies of this problem have not included explicit three-dimensional monomer diffusion, and consequently, missed out on two important aspects: (i) the barrier, even when it is far from the polymers, affects free diffusion of monomers and reduces their adsorption at the tips, while (ii) parallel filaments could interact through the monomer density field ("diffusive coupling"), leading to negative interference between them. In our study, both these effects are included and their consequences investigated in detail. A mathematical treatment based on a set of continuum Fokker-Planck equations for combined filament-wall dynamics suggests that the barrier-induced monomer depletion reduces the growth velocity and also the stall force, while the total force produced by many filaments remains additive. However, Brownian dynamics simulations show that the linear force-number scaling holds only when the filaments are far apart; when they are arranged close together, forming a bundle, sublinear scaling of force with number appears, which could be attributed to diffusive interaction between the growing polymer tips.

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