This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protection of genetic information is extremely important for the function of the cell. The DNA inside the cell nucleus is the blueprint used by the cell to create the host of molecular machinery that carries out all cellular tasks. The nuclear pore complex (NPC) is the first and foremost guard in that protection. It is a very large macromolecular assembly of proteins that sits in the nuclear envelope and determines which cellular materials may pass into and out of the nucleus. The NPC is perhaps the largest protein structure in eukaryotic cells [118], and as a result, it is difficult to study experimentally. Thus, the mechanism by which the NPC selectively allows good material across the nuclear envelope, while preventing the transit of the bad , remains unknown. It is known, however, that in order to cross the nuclear envelope, a large molecule must first associate with a transport receptor protein (reviewed in [118 123]). It is hypothesized that proteins in the NPC recognize the transport receptor and allow the complex to pass. Understanding precisely how this recognition occurs is vital to determining how the NPC protects the nucleus. In order to shed light on the gating mechanism of the NPC, the Resource studied the transport receptor importin-beta in the presence of nuclear pore proteins using a combination of molecular dynamics simulations and bioinformatics. NAMD [44] was used to perform molecular dynamics on the importin-beta in a solution with nuclear pore proteins whose concentration ranged from 50-90 mM. Complete systems averaged roughly 150,000 atoms and were simulated between 20-50 ns each, posing a significant challenge and requiring extremely efficient computing on large (128- 256 processor) machines. In addition, sequences of importin-beta from eight species were aligned, and conserved residues on the importin-beta surface were thus determined and visualized using VMD [49]. During the course of the simulations, the nuclear pore proteins were found to bind to the surface of importin-beta. Binding of proteins near the conserved residues on the surface indicated an increased likelihood that the observed binding is relevant to in vivo NPC recognition of importin-beta. Using this combination of simulations and sequence information, the Resource was able to confirm three of the four importin-beta binding spots which were experimentally known [18 20]. We also predicted five novel binding sites on the importin-beta surface [25]. Furthermore, in a definitive display of the predictive power of computational science, we identified one binding spot independent of and concurrently with experimental researchers, who reported the binding spot [21] only a few months prior to the Resource. The work has prompted further simulations by the Resource on two other transport receptors, NTF2 and cse1p, which will further help to reveal how interactions between the NPC and transport receptors determine the gating mechanism of nuclear transport.
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