Biology subjects

Doubleday, C.

Publications and source records attributed to Doubleday, C..

3 recordsLinked to original sources

bioRxiv2025

The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions

Bacterial non-homologous end joining (NHEJ) is a DNA double-strand break (DSB) repair pathway that relies on the Ku-LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminus has been highlighted for its role in LigD recruitment to DNA DSBs and stimulation of ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences other LigD activities. Here, we combine NMR spectroscopy, structural modelling and mutational analysis to define the interaction interface between the Ku C-terminus and LigD. We identify critical residues in Ku (E246, V248, S258, K260, and N266) and the LigD polymerase (D162, V194, R198) and ligase (D522, K579, L580) domains that mediate this interaction. Functional assays reveal that Ku stimulates LigD ligase activity through contacts with both polymerase and ligase domains, while Ku attenuates template-dependent polymerase activity, contrasting previous studies with Pseudomonas aeruginosa homologs. Disrupting the Ku-LigD interface, either through Ku or LigD mutations, abolishes ligase stimulation and restores polymerase activity, highlighting a dual regulatory mechanism. Our data supports a model where Kus C-terminal region forms a bipartite interface with LigD to balance repair activity. These findings provide mechanistic insight into the Ku-LigD repair mechanism and uncovers species-specific differences in bacterial NHEJ. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/693941v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@802e3org.highwire.dtl.DTLVardef@1f5ac14org.highwire.dtl.DTLVardef@1f46677org.highwire.dtl.DTLVardef@7312b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Kinesin-8 motors dimerize by folding their proximal tail domain into a compact helical bundle

Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal {beta}-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the Candida albicans Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of CaKip3 folds into a compact 92 [A]-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.

biochemistry↗

bioRxiv2022

Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

biochemistry↗