Structure of the complex of a mitotic kinesin with its calcium binding regulator

Department of Biochemistry/Biophysics, University of California, San Francisco, CA 94107, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2009; 106(20):8175-9. DOI: 10.1073/pnas.0811131106
Source: PubMed


Much of the transport, tension, and movement in mitosis depends on kinesins, the ATP-powered microtubule-based motors. We report the crystal structure of a kinesin complex, the mitotic kinesin KCBP bound to its principal regulator KIC. Shown to be a Ca(2+) sensor, KIC works as an allosteric trap. Extensive intermolecular interactions with KIC stabilize kinesin in its ADP-bound conformation. A critical component of the kinesin motile mechanism, called the neck mimic, switches its association from kinesin to KIC, stalling the motor. KIC denies access of the motor to its track by steric interference. Two major features of this regulation, allosteric trapping and steric blocking, are likely to be general for all kinesins.

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    • "Endoplasmic reticulum can also actively pump large amounts of calcium into their luminal spaces, and indeed the specialized cardiac sarcolemma are an unusually well developed case of this organelle. Calcium is involved in the shape change of many cells including immune cells, in both actin and tubulin polymerization and in both kinesin and myosin motoric activity (Sun, Lou et al. 2009; Vinogradova, Malanina et al. 2009). "
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    • "However, a principle difficulty in structural analysis of the kinesin system, as with the other two known cytoskeletal motors (myosin and dynein), has been the inability to obtain atomic-resolution structures of the motor while complexed to the partner filament. To date, the only atomic-resolution structural data available for kinesin comes from X-ray crystal structures of the molecule by itself (Marx et al. 2009) or in complex with a regulatory partner (Vinogradova et al. 2009). However, kinesin’s enzymatic properties change markedly in the absence of microtubules, where for example the ATPase rate drops to a basal level ∼1,000-fold reduced over the motile, microtubule-attached state (Hackney 1988). "
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    ABSTRACT: Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or "heads", to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor's principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin's ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin's ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin-microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.
    Biophysical Reviews 06/2011; 3(2):85-100. DOI:10.1007/s12551-011-0049-4
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    • "A crystal structure of kinesin-14 KCBP, thought to represent the ATP state, shows the C-terminus docked onto the motor core, forming the 'neck mimic', resembling the kinesin-1 neck linker [16]. The KCBP C-terminus performs a different role than the C-terminus of other kinesin-14 motors in regulating Ca+2-calmodulin binding by the motor, which undocks the neck mimic and inhibits motor binding to microtubules in the ADP state [29]. The finding that the Ncd C-terminus resembles the KCBP neck mimic is unexpected, given that there is no apparent inhibitory binding partner for Ncd. "
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    ABSTRACT: 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. 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. 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.
    BMC Structural Biology 07/2010; 10(1):19. DOI:10.1186/1472-6807-10-19 · 1.18 Impact Factor
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