Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer. / Master of Science

Alpha-helical coiled-coils are common protein structural motifs with varied mechanical roles, such as, tropomyosin in muscle contraction or neck-stalks of kinesins and myosins, in motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils both, globally and locally. Normal mode analysis for global elastic properties revealed a buckling instability due to inherently present weak non-bonded forces. We characterized this using a critical buckling length (lc). For coiled-coils, lc was significantly less than their persistence length thereby governing the filament conformation. We also found that mutations to the hydrophobic residues at the knob-into-hole interface affect elasticity of coiled-coils significantly. We built a flexibility map of tropomyosin using a local fluctuation analysis and found regional variations in flexibilities due to such breaks in the knob-into-hole packing. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Actin binding sites in zones and broken core regions due to acidic residues at the hydrophobic face such as, the Asp137 and the Glu218, are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm, respectively. Such variations in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding. Non-claret disjunction (Ncd), is a Kinesin-14 family protein that walks to the microtubule's minus end. Although available structures show its alpha-helical coiled-coil neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck travel is a guided diffusion involving sequential intermediate contacts with the motor head. The post-stroke is at a higher free-energy minimum than the pre-stroke. The importance of intermediate contacts correlates with the existing motility data including those of mutant Ncds and other members of the kinesin-14 family. While the forward motion has a ~4.5 kBT (kB: Boltzmann constant, T = 300 K) free energy barrier, recovery stroke goes nearly downhill in free energy. The hysteresis in forward and reverse neck motion energetics arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key for the directed motility of Ncd. Although it is known that neighboring Ncds on a microtubule (MT) have an attractive interaction and a group of Ncds act cooperatively, the physical basis of neither this attraction nor the cooperativity is known. From structural analysis of Ncd neighbors on an MT lattice we find that steric hindrances between the coiled-coil neck-stalks of longitudinal neighbors drive synchrony among a group of Ncds on a single protofilament. Across lateral dimers, surface loop L2 of the motor-head (MH) that is not bound to the MT (unbound-MH) in a pre-stroke dimer, is seen to have strong attraction to the nucleotide pocket in the MH that is bound to MT (bound-MH) of its off-axis neighbor. Such an attraction will however impede the motility in both the dimers. We hence propose rules that drive motor binding to an MT site in the presence of immediate neighbors such that motility of the group is not compromised. The unbound-MH, whose role in the walking step of an Ncd was unclear, is thus seen to regulate MT decoration.

Kinesins are molecular motors that transport intracellular cargos along microtubules (MTs) and influence the organization and dynamics of the MT cytoskeleton. Their force-generating functions arise from conformational changes in their motor domain as ATP is bound and hydrolyzed, and products are released. In the budding yeast Saccharomyces cerevisiae, the Kar3 kinesin forms heterodimers with one of two non-catalytic kinesin-like proteins, Cik1 and Vik1, which lack the ability to bind ATP, and yet they retain the capacity to bind MTs. Cik1 and Vik1 also influence and respond to the MT-binding and nucleotide states of Kar3, and differentially regulate the functions of Kar3 during yeast mating and mitosis. The mechanism by which Kar3/Cik1 and Kar3/Vik1 dimers operate remains unknown, but has important implications for understanding mechanical coordination between subunits of motor complexes that traverse cytoskeletal tracks. In this study, we show that the opportunistic human fungal pathogen Candida albicans (Ca) harbors a single version of this unique form of heterodimeric kinesin and we present the first in vitro characterization of this motor. Like its budding yeast counterpart, the Vik1-like subunit binds directly to MTs and strengthens the MT-binding affinity of the heterodimer. However, in contrast to ScKar3/Cik1 and ScKar3/Vik1, CaKar3/Vik1 exhibits weaker overall MT-binding affinity and lower ATPase activity. Preliminary investigations using a multiple motor motility assay indicate CaKar3/Vik1 may not be motile. Using a maltose binding protein tagging system, we determined the X-ray crystal structure of the CaKar3 motor domain and observed notable differences in its nucleotide-binding pocket relative to ScKar3 that appear to represent a previously unobserved state of the active site. Together, these studies broaden our knowledge of novel kinesin motor assemblies and shed new light on structurally dynamic regions of Kar3/Vik1-like motor complexes that help mediate mechanical coordination of its subunits. / Thesis (Master, Biochemistry) -- Queen's University, 2012-02-29 17:15:03.654