Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

Abstract

Background: Kar3Vik1 is a heterodimeric kinesin with one catalytic subunit (Kar3) and one noncatalytic subunit (Vik1). Results: Vik1 experiences conformational changes in regions analogous to the force-producing elements in catalytic kinesins. Conclusion: A molecular mechanism by which Kar3 could trigger Vik1's release from microtubules was revealed. Significance: These findings will serve as the prototype for understanding the motile mechanism of kinesin-14 motors in general.It is widely accepted that movement of kinesin motor proteins is accomplished by coupling ATP binding, hydrolysis, and product release to conformational changes in the microtubule-binding and force-generating elements of their motor domain. Therefore, understanding how the Saccharomyces cerevisiae proteins Cik1 and Vik1 are able to function as direct participants in movement of Kar3Cik1 and Kar3Vik1 kinesin complexes presents an interesting challenge given that their motor homology domain (MHD) cannot bind ATP. Our crystal structures of the Vik1 ortholog from Candida glabrata may provide insight into this mechanism by showing that its neck and neck mimic-like element can adopt several different conformations reminiscent of those observed in catalytic kinesins. We found that when the neck is ␣-helical and interacting with the MHD core, the C terminus of CgVik1 docks onto the central ␤-sheet similarly to the ATP-bound form of Ncd. Alternatively, when neck-core interactions are broken, the C terminus is disordered. Mutations designed to impair neck rotation, or some of the neck-MHD interactions, decreased microtubule gliding velocity and steady state ATPase rate of CgKar3Vik1 complexes significantly. These results strongly suggest that neck rotation and neck mimic docking in Vik1 and Cik1 may be a structural mechanism for communication with Kar3.Eukaryotic cells rely on nanometer-sized motors called kinesins to transport cellular components along microtubules (1) or to help build the mitotic spindle and distribute chromosomes between daughter cells (2,3). Recent studies have shown that dynamic interactions between the neck and a short region of either the N or C terminus of the motor domain form a structure responsible for force generation by the neck (4 -7) and that its conformation and interactions with the motor domain core, or regulatory proteins, is linked to the nucleotide-and microtubule-binding state of the motor (8, 9). In kinesin-1, this region forms an N-terminal extension of the motor domain, called the "cover strand" (5), and in kinesin-14 motors this region is at the C terminus, after the ␣6 helix, and has been dubbed the "neck mimic" (8).Kar3 is a kinesin-14 that plays essential roles in mitosis, meiosis, and karyogamy in Saccharomyces cerevisiae and Candida albicans (10 -13). These include cross-linking, stabilizing, and sliding spindle pole microtubules, as well as depolymerizing microtubules (10,14). To accomplish this array of functions, Kar3 associates with two discrete regulatory subunits, Cik1 and Vik1 (14 -16), whose mot...

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…The kinesin-14 motors are found in dimeric form, and, in most species, the majority are homodimers (Fig. 1A), with the exception of several yeast species, for instance the heterodimers Kar3-Cik1 and Kar3-Vik1 in budding yeast (Barrett et al, 2000;Duan et al, 2012) and in Candida glabrata (Joshi et al, 2013).…”

Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation

“…S2 in the supplemental material). The first is that CaCik1 lacks ATPase activity, which is also absent in ScCik1, ScVik1, and C. glabrata Vik1 (20,(62)(63)(64). The second is that CaCik1 lowers the concentration of microtubules required to activate ATP turnover in the CaKar3 subunit.…”

“…As this affinity was also higher than that of the monomeric ADP-bound CaKar3MD construct, it appears that CaCik1 may tether the CaKar3/Cik1 complex to the microtubules in the ADP-bound state of CaKar3. If the models proposed for motility of other Kar3/Cik1 and Kar3/Vik1 motors are relevant to CaKar3/Cik1, conversion to the ATP-bound state presumably reverses this interaction toward a CaKar3-binding mode (20,(62)(63)(64).…”

Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s

“…In contrast to the microtubule (MT) plus-end directed processive kinesins, kinesin-14s are not processive as single molecules; they promote MT minus-enddirected force and use an ATP-promoted powerstroke to crosslink and slide one MT relative to another (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Sequence analysis indicates that all members of the kinesin-14 subfamily are dimeric, yet the structural organization of kinesin-14 motors differs from the N-terminal processive kinesins.…”

“…The kinesin-14s exhibit C-terminal motor domains connected by an N-terminal continuous coiled-coil stalk with an N-terminal ATP-independent MT binding site (7,(18)(19)(20)(21)(22). And although most of the kinesin-14s are homodimeric, some yeast species, including Saccharomyces cerevisiae and Candida glabrata, contain heterodimeric kinesin-14s (13,14,19). The conventional hypothesis for homodimeric kinesin-14 force generation proposed that only one motor head interacts with the MT and only one ATP turnover is required to complete the powerstroke (Fig.…”