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Biochemical characterization of the Nup62⋅58⋅54 nucleoporin complex and mutational analysis of the exportin CRM1

dc.contributor.advisorGörlich, Dirk Prof. Dr.
dc.contributor.authorChug, Hema
dc.titleBiochemical characterization of the Nup62⋅58⋅54 nucleoporin complex and mutational analysis of the exportin CRM1de
dc.contributor.refereeLührmann, Reinhard Prof. Dr.
dc.description.abstractengNuclear pore complexes (NPCs) are giant molecular machines embedded in the double membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleo-cytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins (Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer. Nup62·58·54 subcomplex is a nucleoporin subcomplex in the NPC’s central channel, the molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeat- motifs.  The C-terminal half is structured into coiled-coil domains that engage in tight protein-protein interactions to hold the complex together and to anchor it to the NPC scaffold. In this project, I aimed to elucidate the molecular structure of the X.laevis ∆FG-Nup62·58·54 complex by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as Nup54, in bacterial expression has been a challenge towards obtaining large amounts of nucleoporins required for crystallographic analysis. We established a system where all three subunits of xl∆FGNup62·58·54 were co-expressed together in E.coli. The soluble amount of Nup54 was greatly enhanced, when co-expressed together with its interaction partners. Biochemical analysis of the recombinant ∆FG-Nup62·58·54 complex revealed a monomeric, non-spherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also, in contrast to previously published reports, the ∆FG-Nup62·58·54 complex did not show any tendency to form protein-concentration dependent higher-order oligomers. However, extensive crystallization trials failed to crystallize ∆FG-Nup62·58·54 complex, probably due to inherent flexibility in its structure. Therefore, we generated and screened a series of single-domain antibodies (nanobodies, Nbs) against ∆FG-Nup62·58·54 complex and identified a few that stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric interactions. Crystallization trials of ∆FG-xlNup62·58·54 complex conjugated to one such nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction potential to ∼7.5Å.  Thus, nanobody conjugation of the ∆FG-xlNup62·58·54 complex aided in its crystallization and can be used as a general approach to stabilize NPC subcomplexes. Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62·58·54 complex is thought to recruit the Nup62·58·54 complex to the NPCs. This interaction in our experiments, however, turned out to be surprisingly weak. This now predicts additional interaction sites for a high avidity anchorage of the Nup62·58·54 complex to the NPC scaffold.In a parallel project, we aimed to obtain insights into exportin CRM1’s conformational states by analyzing its sequence conservation in a large number of diverse species, starting from lower eukaryotes such as Trichomonas till humans. Individual point mutations in 21 absolutely conserved CRM1 residues produced interesting phenotypes with either greatly enhanced or weakened cargo binding, suggesting that many of these evolutionary conserved residues are critical for stabilizing distinct conformational states of cargo-free or cargo-bound CRM1. These mutants will be exploited further in structural studies that explore the conformation space available to
dc.contributor.coRefereeRehling, Peter Prof. Dr.
dc.subject.engX-ray Crystallographyde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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