A Structural Analysis of the Interaction between ncd Tail and Tubulin Protofilaments

Wendt, Thomas; Karabay, Arzu; Krebs, Angelika; Gross, Heinz; Walker, R. A.; Hoenger, Andreas

doi:10.1016/j.jmb.2003.08.051

Cited by 20 publications

(18 citation statements)

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“…These unique properties include their direction of movement towards the minus-end of microtubules, and their reversed building plan that places the motor domain at the C-terminal end of the polypeptide chain (Walker et al, 1990; McDonald et al, 1990; Endow et al, 1994; Mountain et al, 1999). In addition, all kinesin-14’s studied thus far are non-processive motors (Case et al, 1997; de Castro et al, 2000; Foster and Gilbert, 2000; Fink et al, 2009), and for all of them the cargo-binding site interacts with a second microtubule, but in a non-motor like fashion and without the control of nucleotide state (Karabay and Walker, 1999; Wendt et al, 2003). Hence, unlike kinesin-1 members or other highly processive kinesins, kinesin-14s would be very ineffective motors for long-distance transport.…”

Section: Discussionmentioning

confidence: 99%

“…Ncd is required for proper chromosome distribution in meiosis and early mitosis and has been implicated in having roles in bipolar spindle assembly and its maintenance as well as spindle pole formation (Endow et al, 1994; Oladipo et al, 2007). Ncd uses its C-terminal motor domains and N-terminal non-motor microtubule binding domains (Karabay & Walker, 1999; Wendt et al, 2003) to crosslink antiparallel microtubules within the spindle midzone and parallel microtubules at the spindle poles.…”

Section: Introductionmentioning

confidence: 99%

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Common mechanistic themes for the powerstroke of kinesin-14 motors

González

Cope

Rank

et al. 2013

Journal of Structural Biology

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Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Common mechanistic themes for the powerstroke of kinesin-14 motors

González

Cope

Rank

et al. 2013

Journal of Structural Biology

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“…The yeast Kar3 was the first motor proposed to have an N-terminus that can bind independently to microtubules, based on localization studies [Meluh and Rose, 1990]. The presence of a second nucleotide-insensitive microtubule domain has also been identified in Kinesin-1, -7, -8, -10, and -14 [Navone et al, 1992; Yen et al, 1992; Karabay and Walker, 1999; Shiroguchi et al, 2003; Wendt et al, 2003; Seeger and Rice, 2010; Moua et al, 2011]. The microtubule binding properties of the non-motor regions differ from those of the motor domains such that they are likely to provide a second low affinity binding site for the kinesin to increase its affinity for microtubules.…”

Section: Microtubule Interactions With Kinesin Non-motor Regionsmentioning

confidence: 99%

“…The N-terminal domain can bind to and bundle microtubules in vitro [Chandra et al, 1993]. In fact, the N-terminal 200 amino acid tail contains two separate microtubule binding sites that are ATP-insensitive [Karabay and Walker, 1999; Wendt et al, 2003]. The mammalian, S. cerevisiae , and S. pombe Kinesin-14 counterparts HSET, Kar3, and Klp2 also have N-terminal microtubule binding domains in addition to the C-terminal motor domain [Meluh and Rose, 1990; Ando et al, 1994; Kuriyama et al, 1995; Braun et al, 2009].…”

Section: Microtubule Interactions With Kinesin Non-motor Regionsmentioning

confidence: 99%

The molecular basis for kinesin functional specificity during mitosis

Welburn

2013

Cytoskeleton

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Microtubule-based motor proteins play key roles during mitosis to assemble the bipolar spindle, define the cell division axis, and align and segregate the chromosomes. The majority of mitotic motors are members of the kinesin superfamily. Despite sharing a conserved catalytic core, each kinesin has distinct functions and localization, and is uniquely regulated in time and space. These distinct behaviors and functional specificity are generated by variations in the enzymatic domain as well as the non-conserved regions outside of the kinesin motor domain and the stalk. These flanking regions can directly modulate the properties of the kinesin motor through dimerization or self-interactions, and can associate with extrinsic factors, such as microtubule or DNA binding proteins, to provide additional functional properties. This review discusses the recently identified molecular mechanisms that explain how the control and functional specification of mitotic kinesins is achieved. © 2013 Wiley Periodicals, Inc.

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“…Ncd has two identical heads, located at the C termini of the polypeptides, which are tightly attached to a superhelical segment. The N-terminal tail contains also a secondary ATP-independent MT-binding site (11,12). Although many lines of experimental evidence support the view that Ncd is a nonprocessive motor, it was reported recently that full-length Ncd may exhibit some characteristics of a processive protein (13).…”

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confidence: 99%

Interactions between Subunits in Heterodimeric Ncd Molecules

Kocik

Skowronek

Kasprzak

2009

Journal of Biological Chemistry

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The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a singleheaded molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5-adenylyl-␤,␥-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

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A Structural Analysis of the Interaction between ncd Tail and Tubulin Protofilaments

Cited by 20 publications

References 31 publications

Common mechanistic themes for the powerstroke of kinesin-14 motors

Common mechanistic themes for the powerstroke of kinesin-14 motors

The molecular basis for kinesin functional specificity during mitosis

Interactions between Subunits in Heterodimeric Ncd Molecules

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