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MECHANISM OF PINK1-MEDIATED UBIQUITIN PHOSPHORYLATION 10.17863/CAM.24555 Schubert, Alexander Fabian 0000000218343787 Komander, David 0000000280924320 Ubiquitin phosphorylation by PINK1 (PTEN-induced Putative Kinase 1) is crucial for mitochondrial quality control and loss or mutation of PINK1 can lead to autosomal recessive juvenile parkinsonism (AR-JP). PINK1 is an unusual kinase, as it is characterised by three unique insertions in its kinase N lobe and a C-terminal region after the kinase domain. Despite great effort, a structure of PINK1 could not be determined and the molecular mechanism of ubiquitin phosphorylation and the effect of the PINK1 AR-JP patient mutations remained elusive. The versatile modifier ubiquitin (Ub) is also an unusual kinase substrate, as its phosphorylation site (Ser65) is not exposed, but protected by the Ub fold. Hence, it was not clear how a kinase would be able to target Ser65 of Ub. This work shows that Ub needs to adopt a previously described conformation in order to be efficiently phosphorylated by PINK1. NMR experiments revealed that in a small population of Ub the last β-strand is retracted, resulting in a more accessible Ser65 loop. It could be shown that PINK1 binds the Ser65 loop in this C-terminally retracted conformation (Ub-CR), but not in the ‘common’ conformation. In addition, it could be shown that Ub trapped in the Ub-CR conformation by point mutations (Ub TVLN) is phosphorylated significantly faster than Ub wt, which only adopts the Ub-CR conformation at very low frequency. To further elucidate how PINK1 binds and phosphorylates Ub, the kinase domain of Pediculus humanus corporis (Ph)PINK1 was crystallised in complex with Ub TVLN stabilised by a nanobody. The structure revealed many peculiarities of PINK1, such as the architecture of the unique insertions and the C-terminal region. Together with NMR and mass spectrometry studies, the structure explains how PINK1 interacts with ubiquitin via insertion-3 and its activation segment, and how PINK1 utilises the Ub- CR conformation for efficient Ser65 phosphorylation. In addition, the structure shows that two autophosphorylation sites in the N lobe regulate PINK1, by stabilising the functionally important insertions. The structure helped our understanding of the molecular basis of over 40 AR-JP patient mutations and may guide the design of ARJP therapeutics in the future. University of Cambridge 2018-07-20 Thesis Doctoral Doctor of Philosophy (PhD) en MRC Research Studentship program https://www.repository.cam.ac.uk/handle/1810/277271 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/309777c8-1df1-46d8-9ace-21813d7970c3/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b9272310-480c-4ef7-840b-1741fa48454c/download 4e2616df1f5ffdcae289a48a62b83141 https://www.rioxx.net/licenses/all-rights-reserved/ PINK1 Kinase Parkinson's disease Structural biology Ubiquitin Mitophagy