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Hancock Lab

Molecular Biomechanics

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Kinesin Mechanochemistry

We are interested in uncovering how different kinesin motor proteins are optimized for their cellular tasks. Because the kinesin walking mechanism involves two motor domains working in a coordinated manner, we have focused our efforts on understanding the Kinesin-2 family of motors that contain two distinct motor domains instead of the normal homodimer arrangement.  As these motors are necessary for the formation of cilia and flagella, the study of Kinesin-2 motors impacts a wide range of important cell biological questions and pathologies including developmental defects, polycystic kidney disease, and defects in flagellar motility. 
We have generated a number of mutant and chimaeric Kinesin-2 motors, and we are using single-molecule fluorescence experiments, optical tweezer studies, biochemical assays and computational modeling to understand the chemomechanical cycle underlying Kinesin-2 walking.  See Motility Assay page for a description of some of these assays.

The Kinesin neck linker domain

Mechanics of the Kinesin Neck Linker Domain.  The neck linker domain connects each head to the coiled coil and serves three important functions as described.  Diverse kinesins have neck linkers ranging from 14 to 18 amino acids, which is predicted to alter the transmission of force between the two motor domains.

Recently, this work has focused on the role of the flexible neck linker domain, a sequence of 14-18 amino acids that connects the catalytic motor domain to the coiled-coil rod domain.  We found that the ability of Kinesin-2 as well as other diverse kinesins to walk long distances without detaching (motor processivity) is regulated by the length of the neck linker domain (1-3).  We are currently focusing our efforts on understanding the precise biochemical steps in the hydrolysis cycle that are altered by inter-head tension.

Gating and stepping of kinesin-2. We are focusing on understanding specific mechanochemical tuning of kinesin-2 motors by carrying out stopped-flow kinetics and optical trapping experiments (in collaboration with the Block lab).  In our recent optical tweezer work, we found that the processivity of kinesin-2 is strongly dependent on external load – whereas kinesin-1 is able to maintain processivity under load, kinesin-2 run lengths are strongly diminished against external loads (5).  In examining the gating mechanisms that regulate kinesin-2 processivity, we found that the front-head gating paradigm that is thought to play an important role in kinesin-1 processivity is not present in the kinesin-2 hydrolysis cycle.  Specifically, when the motor is in a two-head-bound state, ATP can freely bind to the leading head (6).  This study also showed that kinesin-2 motors in their low affinity ADP state detach relatively slowly from microtubules and suggest that kinesin-2 spends a large fraction of its ATP hydrolysis cycle in a low affinity state.  This biochemical result provides an explanation for the optical trapping result – kinesin-2 resides in a weak binding state for much of its hydrolysis cycle but this state detaches relatively slowly in the absence of load, allowing the motor to achieve substantial unloaded processivity.  However, in the presence of external load, the motor detaches readily from this state resulting in short run lengths when stepping against external loads.  We are currently studying the implications of this result for bidirectional transport.

 

References:

1. Muthukrishnan, G., Zhang, Y., Shastry, S., and Hancock, W.O. (2009). The processivity of kinesin-2 motors suggests diminished front-head gating. Current Biology 19(5), 442-447. Supplemental Data.
2. Shastry, S., and Hancock, W.O. (2010). Neck linker length determines the degree of processivity in Kinesin-1 and Kinesin-2 motors. Current Biology 20, 939-943. Supplemental Data.
3. Shastry, S., and Hancock, W.O. (2011). Interhead tension determines processivity across diverse N-terminal kinesins. Proc Natl Acad Sci U S A 108, 16253-16258.
4. Kinesin processivity is gated by phosphate release.  Milic B., Andreasson J.O., Hancock W.O., Block S.M.  2014.  Proc Natl Acad Sci USA. 111(39):14136-40
5. Processivity of the kinesin-2 KIF3A/B results from rear head gating and not front head gating.  G.Y. Chen, D.F. Arginteanu and W.O. Hancock. 2015. J. Biol Chem. 290(16):10274-94.  PMID: 25657001
6. Andreasson, J.O., S. Shastry, W.O. Hancock, and S.M. Block.  The mechanochemical cycle of mammalian kinesin-2 KIF3A/B under load. 2015.  Current Biology. 25(9):1166-1179.  PMID: 25866395
7. Andreasson, J.O., B. Milic, W.O. Hancock, and S.M. Block.  Examining kinesin processivity within a general gating framework. 2015.  Elife.

 

Other Research Topics:

Kinesin Mechanochemistry
Chemomechanical Modeling
Nanobiotechnology and Microscale Transport
Artificial Mitotic Spindle
Microtubule Polarity in Neurons
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