Kinesin-12, Kif15 is a molecular motor involved in bipolar spindle assembly. Kif15 function is regulated through autoinhibition of its C-terminal tail and binding to the microtubule-associated protein Tpx2. Previous studies have reported Kif15 to function as a tetramer as well as a dimer. In this study, a cross-linking mass spectrometry (XL-MS) protocol and analysis workflow was developed to study the structural organisation of Kif15. Using XL-MS studies, it was found that Kif15 adopts a parallel tetramer conformation, which is autoinhibited by its C-terminal leucine zipper. Next, we show that this autoinhibited conformation is stabilised by the binding partner Tpx2. We also show that there is a shift in the binding interface between Kif15 and Tpx2 when microtubules are present and absent. In the presence of microtubules, Tpx2 mainly binds to the leucine-zipper of the Kif15 motor, whereas in the absence of the microtubules, this binding is exclusively localised to the fourth coiled-coil. We also reveal that Tpx2 adopts a dimeric conformation at physiological ionic strength. Finally, to understand the function of Kif15 in-vivo, we have developed putative Kif15 knock-out cell lines and developed a cross-linking protocol to cross-link Kif15 in cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:767116 |
| Date | January 2018 |
| Creators | Hussain, Hamdi |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/111537/ |
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