Assembly and Structure of the Nuclear Pore Complex
The nuclear pore complex (NPC) is a massive (120 megadaltons [MDa] in vertebrates) supramolecular assembly composed of ~30 different proteins (nucleoporins or nups) that are organized into several subcomplexes. Because of its high symmetry, the NPC contains multiple copies of each subcomplex, thus accounting for ~1,000 protein molecules in the vertebrate NPC. As the sole mediator of macromolecular transport across the nuclear envelope, the NPC adopts a pivotal role in cellular physiology, which becomes evident in many oncogenic and developmental defects caused by impaired NPC function. Although fundamental aspects of the NPC architecture have been known for decades, its detailed structure has largely remained elusive, primarily as a result of heterogeneity of isolated, purified NPCs. Therefore, our research group has embarked on an alternative approach, which ultimately aims to build up the NPC from single components to arrive at a well-defined state. Because the assembly of the entire NPC is a formidable task, we have thus far focused on the structural and functional characterization of various subcomplexes, including the yeast heptameric Nup84 complex, which is a key building block of the NPC. Our crystallographic studies suggest that multiple copies of this subcomplex cluster in a specific way, forming a coat for the nuclear pore membrane. Using recombinant proteins, we are currently assembling this subcomplex in vitro and pursuing crystallographic studies to determine its three-dimensional (3D) structure. We are also working on the central channel subclomplex composed of the three nucleoporins, p62, Nup54, and Nup58. Our structural and biophysical data have elucidated a novel stoichiometry for this subcomplex nups and led us to postulate the "ring cycle" hypothesis where sliding of alpha helical segments of these nups allows for large diameter changes in the central channel coupled to karyopherin occupancy of their FG-repeat domains. We expect our data to provide profound insights into the principles of NPC assembly, structure, and function and hence contribute to a better understanding of NPC-related diseases. Students will be exposed to various stages of the pipeline from gene to a 3D protein structure, including recombinant protein expression in bacteria, protein purification, biochemical analysis, crystallization, and structure determination.