| Nuclear pore complexes (NPCs) act as the gatekeepers between the cytoplasmic and nucleoplasmic compartments of eukaryotic cells. In order to gain insight into mechanisms of nucleocytoplasmic transport as well as the nature of protein-protein interactions in the NPC, I have taken a structural approach to studying its components. This work focuses on Nup116p, a yeast nucleoporin that plays an important role in the terminal steps of mRNA export.; I have determined the solution structure of the C-terminal ∼150 residues of Nup116p, the domain responsible for targeting the protein to the NPC. The structure consists of a large beta sheet sandwiched against a smaller one, flanked on both sides by alpha-helical stretches, similar to the structure of the human homologue hNup98. In unliganded form, Nup116p-C exhibits evidence of exchange among multiple conformations, raising the intriguing possibility that it may adopt distinct conformations when bound to different partners in the NPC.; I have also shown that a peptide from the N terminus of the nucleoporin Nup145p-C binds Nup116p-C. This previously unknown interaction may explain the unusual asymmetric localization pattern of Nup116p in the NPC. Strikingly, whereas HSQC spectra of unbound Nup116p-C indicate conformational exchange, this exchange phenomenon disappears or is greatly reduced in corresponding spectra of peptide-bound Nup116p-C, suggesting that the presence of a binding partner stabilizes the domain's conformation. Additionally, I have identified a short region of Nup82p that bears sequence similarity to the Nup145p-C peptide and exhibits weak binding to Nup116p-C. Chemical shift mapping suggests that Nup116p-C uses the same binding site to interact with both Nup145p-C and Nup82p. These studies offer the first high-resolution glimpse of a yeast nucleoporin structural domain and may provide insights into NPC architecture and function. |
