A kinesin motor in a force-producing conformation.
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2010-07-05
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BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
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Heuston, Elisabeth, C Eric Bronner, F Jon Kull and Sharyn A Endow (2010). A kinesin motor in a force-producing conformation. BMC Struct Biol, 10. p. 19. 10.1186/1472-6807-10-19 Retrieved from https://hdl.handle.net/10161/4362.
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Scholars@Duke
Sharyn Anne Endow
Research in my laboratory focuses on the mechanisms that ensure proper chromosome transmission in dividing cells. My laboratory's studies have contributed to the identification of molecular motor proteins as the force-generating proteins underlying spindle and chromosome movements during cell division. Much of our current efforts is directed towards understanding the molecular basis of motor force production and the contribution of forces produced by motor proteins to spindle and chromosome dynamics in living cells.
My laboratory has used molecular genetics and biophysical assays to determine the basis of the reversed directionality of the Ncd motor protein, discovered in my laboratory, compared to the first discovered kinesin microtubule motor protein. Ncd is required for spindle assembly and chromosome division in Drosophila oocytes and early embryos. We showed previously that Ncd moves on microtubules in the opposite direction as kinesin. By constructing chimeric Ncd-kinesin motor proteins, we identified residues that are required for the reversed movement of Ncd. We then mutated single amino acids and made Ncd motors that move in both directions on microtubules. We showed, by analyzing one mutant using biophysical assays, that the mutant motor produced a conformational change in either direction, but the wild-type motor conformational change was biased towards the microtubule minus end. The assays identified residues that are required for motor directionality and explained how motor directionality is determined.
Current studies in my laboratory focus on motor force-generating mechanisms. We are especially interested in defining the conformational changes in the kinesin motor proteins that produce force. Major questions that we are addressing are the following:
- How do kinesin motor proteins use ATP to produce force?
- What residues can be altered to increase force produced by the motors?
- What are the forces across the motors during assembly and division?
We are using structure/function methods together with genetically encoded molecular tension sensors to address these questions. The findings are relevant to the understanding of basic cellular processes, including cell division and transport, and their mechanisms in live cells. Abnormalities in these basic cellular processes are a major cause of abnormalities in dividing cells that are associated with cancers and other human diseases.
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