A kinesin motor in a force-producing conformation.

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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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Sharyn Anne Endow

Professor of Cell Biology

Research in my laboratory focuses on spindle and chromosome dynamics and the mechanisms that ensure proper chromosome transmission and inheritance in dividing cells. Work in my laboratory and others over the past 5-10 years has identified molecular motor proteins as the force-generating proteins underlying movements of the spindle and chromosomes during cell division. Much of our current effort is directed towards understanding the mechanism of motor function, including the molecular basis of motor directionality, and the contribution of motor proteins to spindle and chromosome dynamics in living cells.

During the past several years, we have used molecular genetics to determine the basis of the reversed directionality compared to kinesin of the Ncd motor protein, discovered in my laboratory. Ncd is a microtubule motor that is required for proper spindle assembly in oocytes and early embryos of Drosophila. We showed previously that Ncd moves on microtubules in the opposite direction as kinesin, the founding member of the protein family to which Ncd belongs. By constructing and mutating chimeric Ncd-kinesin motor proteins, we have recently identified residues that are required for the reversed movement of Ncd. We mutated single amino acid residues of Ncd and made motors that move in both directions on microtubules. Analysis of the mutant motors showed that the motors were functional, but directionality was defective. We analyzed one of the mutants using biophysical methods and detected a conformational change which occurred in either direction in the mutant motor, but was biased towards the minus end in the wild-type motor, and occurs upon binding of the motor to the microtubule. These results explain the minus-end movement of Ncd by identifying residues that are required for motor directionality and explaining how the residues impose directionality on the motor.

Our present studies focus on motor directionality and processivity, and mechanisms underlying chromosome distribution in meiosis and mitosis. We are carrying out further studies on the molecular basis of motor directionality and processivity, and the conformational changes the motors undergo during ATP hydrolysis. Studies of selected Ncd mutants are being performed in live cells to determine the effect of altering specific motor functions on the cellular function of the motor. Mutant ncd-gfp gene fusions are constructed for these studies and the GFP is imaged in live oocytes & embryos by laser scanning confocal microscopy. Assays are being developed to analyze the biophysics of specific motor mutants in vivo by live imaging in order to determine the contributions of motors and microtubule dynamics to spindle dynamics, and to correlate these results with the genetic effects of the mutants. These studies should provide new information about the forces that are needed for spindle assembly in living cells and the effects of mutant motors on spindle dynamics and chromosome distribution. Abnormalities in these basic cellular processes are a major cause of somatic abnormalities in mitotically dividing cells and may contribute causally to cellular transformation.

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