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. Our 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.
Our first aim i s 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 i s 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077537-04
Application #
7763210
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Flicker, Paula F
Project Start
2007-02-01
Project End
2012-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
4
Fiscal Year
2010
Total Cost
$274,874
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Apelt, Luise; Knockenhauer, Kevin E; Leksa, Nina C et al. (2016) Systematic Protein-Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex. Mol Cell Proteomics 15:2594-606
Schwartz, Thomas U (2016) The Structure Inventory of the Nuclear Pore Complex. J Mol Biol 428:1986-2000
Knockenhauer, Kevin E; Schwartz, Thomas U (2016) The Nuclear Pore Complex as a Flexible and Dynamic Gate. Cell 164:1162-1171
Kabachinski, Greg; Schwartz, Thomas U (2015) The nuclear pore complex--structure and function at a glance. J Cell Sci 128:423-9
Kelley, Kotaro; Knockenhauer, Kevin E; Kabachinski, Greg et al. (2015) Atomic structure of the Y complex of the nuclear pore. Nat Struct Mol Biol 22:425-431
Berchowitz, Luke E; Kabachinski, Greg; Walker, Margaret R et al. (2015) Regulated Formation of an Amyloid-like Translational Repressor Governs Gametogenesis. Cell 163:406-18
Ulrich, Alexander; Partridge, James R; Schwartz, Thomas U (2014) The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution. Mol Biol Cell 25:1484-92
Andersen, Kasper R; Leksa, Nina C; Schwartz, Thomas U (2013) Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81:1857-61
Bilokapic, Silvija; Schwartz, Thomas U (2013) Structural and functional studies of the 252 kDa nucleoporin ELYS reveal distinct roles for its three tethered domains. Structure 21:572-80
Schwartz, Thomas (2013) Functional insights from studies on the structure of the nuclear pore and coat protein complexes. Cold Spring Harb Perspect Biol 5:

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