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FG-Nucleoporins and the nucleocytoplasmic transport; two distinct molecular mechanisms of multivalent interactions

Valle Aramburu, Iker

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Abstract

FG-Nucleoporins (FG-Nups) are intrinsically disordered proteins (IDPs) located at the nuclear pore complex (NPC) where they form the permeability barrier of the NPC. The selective transport of cargoes, with a molecular weight above 40 kDa (>4 nm), across the NPC is mediated by nuclear transport receptors (NTRs). NTR mediated nucleocytoplasmic transport requires the direct interaction between NTRs and the phenylalanine-glycine motifs (FG-motifs) present in the FG-Nups of the NPC barrier. The selective crossing of NTRs through the ~30 nm permeability barrier of NPC has been shown to occur on the millisecond timescale. However, these fast transport times don’t seem to correlate with the high specificities reported for several FG-Nup/NTR complexes, which are often associated with long lasting complexes. Thus, the understanding of the fast and selective nucleocytoplasmic transport resides in deciphering the molecular basis of the interaction between FG-Nups and NTRs. In my PhD thesis I have focused on understanding the binding mechanism between FG-Nups and NTRs by studying the structure, dynamics and kinetics of multiple FG-Nups upon binding to NTRs. I have shown, with different biophysical techniques like; multiparameter single-molecule fluorescence resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS) and stopped-flow spectroscopy that many FG-Nups are able to bind NTRs forming highly dynamic complexes. Moreover, most FG-Nups engage with different NTRs without undergoing a conformational which I have shown that it is linked to diffusion limited binding not driven by long-range electrostatic interactions. The work presented in this thesis contributed to the proposal of a model where FG-Nups interact with multiple minimalistic low affinity binding motifs with different NTR binding pockets with ultrafast kinetics allowing the fast NPC crossing of NTRs. This novel binding mechanism is due to the high dynamics of IDPs and can also explain why IDPs have enriched the development of higher organisms. Exceptionally, Nup214FG which is not forming part of the central FG-Nup barrier binds to NTRs like CRM1 or Importinβ with a different binding mechanism, where the binding is coupled to a conformational change. Moreover, these different FG-Nup/NTR binding mechanisms don’t seem to be affected by the glycosylation of FG-Nups, which is a highly abundant posttranslational modification in the metazoan FG-Nups located at the NPC. In addition, motivated by the possibility of translating the in vitro results to a physiological environment and be able to study the structure of FG-Nups in situ at the NPC, I am developing a strategy that would improve the study of the structure and dynamics of the disordered regions of the FG-Nups in cells, paving the way toward the intracellular study of IDPs.

Item Type: Dissertation
Supervisor: Melchior, Prof. Dr. Frauke
Date of thesis defense: 27 November 2017
Date Deposited: 20 Jun 2018 08:53
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
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