The objective of this work is to investigate the structures of two nucleotide binding proteins: mevalonate kinase (MVK) and FtsZ. MVK is the key enzyme involved in terpenoid biosynthesis. In this study, we solved the crystal structures of the M. jannaschii MVK apoprotein, as well as the protein in complex with ligands. Its fold was analyzed and firmly established within the GHMP kinase family, in which homoserine kinase (HSK), phosphomevalonate kinase and galactokinase also belong. Structural analysis in combination with enzyme kinetics studies revealed the mechanism of this enzyme upon substrate binding, catalysis and inhibition. In particular, the phosphate-binding loop was found to be critically involved in the binding of nucleotides and terpenoids, via the interaction with a di-phosphate moiety from the ligand. An enzymatic reaction mechanism was constructed based on our structural data and it is consistent with kinetics studies from the literature. In this mechanism, the invariant residue Asp 155 functions as a general base that increases the nucleophilicity of the phosphoryl acceptor. Finally, a virtual screening study has been performed to explore the ligand binding potential of MVK. Compounds predicted to bind MVK were tested and analyzed. FtsZ is a prokaryotic homologue of tubulin that forms the apparatus for bacterial cell division. The structure of a crystal filament of the M. tuberculosis FtsZ complexed with GDP was described in this study. It shows an anti-parallel, left-handed double helical architecture. Compared with the straight crystal filament revealed earlier by other groups, the catalytic T7 loop in our structure is found to be outside the nucleotide binding site, indicating the GTPase is inactive. Furthermore, the buried surface area in our crystal filament is less, probably suggesting the helical FtsZ filament is less stable. We therefore proposed that the hydrolysis of GTP and the releasing of the γ-phosphate group will trigger the rearrangement of the FtsZ fibler, characterized by the exclusion of the T7 loop, which might lead to a less stable helical filament and would be the first step for the disassembly of FtsZ polymer.

Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3. Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique. The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction). Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.

Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3. Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique. The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction). Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.