High Resolution Assembly Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC), a 60-80 MDa modular protein assembly, serves as the exclusive gateway to the nucleus of the cell. In a typical human cell, several thousand NPCs are embedded in the nuclear envelope (NE), the double-layered membrane that wraps the nucleus. Since transcription and translation are spatially divided between nucleus and cytoplasm, NPCs have to fulfill an enormous transport task, mainly consisting of exporting mRNA and ribosomal subunits from the nucleus and importing nuclear pro- teins such as transcription factors. Apart from their regular function, however, the NPC and its constituents, nucleoporins, also play a particularly prominent role in human pathology. Individual nucleoporins are implicated in several types of leukemia and severe liver disease, primary biliary cirrhosis. Furthermore, and most pertinent to this proposal, many pathogenic viruses, including HIV and hepatitis B virus, use the NPC as their entry site to the nucleus. Several viruses also encode proteins, which interrupt nuclear transport of specific substrates as part of their strategy to hijack the cell. In order to fully understand these viral pro- cesses and consequently be able to disrupt viral interaction with the NPC, it is highly desirable to decipher its structure in atomic detail. Such information will provide a basis for targeted drug development against viral pathogens, which is our long-term objective. Due to the complexity and size of the NPC, we propose a two-pronged approach using X-ray crystallographic and electron-microscopic techniques.

This proposal is based on the observation that the NPC is a highly modular assembly composed of individual subcomplexes that arrange along two- and eight-fold rotational symmetries. In vertebrates, NPCs disassemble into these subcomplexes during cell division and reassemble from them afterwards. Our hypothesis is that these sub- complexes are amenable to X-ray crystallographic analysis. The architectural core structure of the vertebrate NPC is essentially composed of three subassemblies, well-characterized heterotrimeric p62 and heterononameric Nup160 complexes and in addition the less-characterized Nup205 complex.

The first aim is to solve the crystal structure of the p62 subcomplex. Its core structure is predicted to be a helical assembly of coiled coils, the arrangement of which the crystal structure will unravel. This structure will provide a basis for understanding the inner interaction network of the NPC assembly. Our second aim is to solve the structure of nonameric Nup160 complex, which is considered to be the platform that later recruits accessory components to the NPC. Importantly, it can be reconstituted in wfrofrom heterodimeric subunits and is then amenable to X-ray crystallography. Structural information gained from our experiments should provide de- tailed molecular insight into construction of a large macromoloecular machine like the NPC and should fur- ther guide rational design of drugs that, for example, specifically disrupt docking of viral capsids to the NPC.