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. The nucleus of the eukaryotic cell is of central importance to an organism. It servesto store and organize genetic material, while separating and protecting this veryimportant information from other cellular components. While the nucleus requiresthis protective isolation, it also needs to communicate with the rest of the cell,exchanging proteins and RNAs, for a variety of nuclear and cytoplasmic processeswhich act in concert. The nuclear pore complex (NPC) is the gatekeeper of the nucleus(www.ks.uiuc.edu/Research/npc/). The NPC central channel, throughwhich all cargo is transported, is filled with unstructured proteins, commonly calledFG-nups. Because of the unstructured nature of these FG-nups, however, pointedexperimental study has been difficult, and as a result, the mechanism by which theNPC selectively allows transport of only certain material remains unknown. It isknown, however, that in order to cross the nuclear envelope, a large molecule mustfirst associate with a transport receptor protein (reviewed in [1?6]), which can bindto FG-nups through hydrophobic binding spots [7?13]. Understanding preciselyhow the FG-nups function as the selective barrier is therefore vital to determininghow the NPC protects the nucleus.In order to explore the selective barrier of an NPC, the Resource has computationallysampled a representative volume of the FG-nup-filled NPC central channel [14].One FG-nup, namely yeast nsp1, was divided into 25 segments, each containing 100amino acids. The resulting 25 segments were then tethered onto a planar surface,forming a 5 by 5 array. The resulting system contains more than 1 million atoms andneeded to be simulated for several microseconds, which is not feasible through allatom(AA) molecular dynamics based on current supercomputer abilities. In orderto overcome this problem, a coarse-grained (CG) model [15,16] was used to extendthe simulation timescale to microseconds and AA simulations were then performedto refine the resulting CG structures. VMD [17] was used to transform the systembetween AA and CG representations and NAMD [18] was used to perform both theAA and CG simulations. By combining CG and AA molecular dynamics, individualnsp1 segments and arrays of them were simulated for as long as 4 microseconds. Thesimulations suggest a bundle-based brush-like structure for the NPC selective barrier:(i) on their surface the brush bundles are dotted with spots of amino acid pairs,phenylalanine and glycine, that are known from both simulations and experimentsto interact favorably with transport receptor proteins [7?13];(ii) the brush bundlesare also interconnected, as FG-nup segments frequently switch from one bundle toanother. Based on the above observations, it appears then that the FG-nups forman energetically favorable environment for transport receptor proteins and that thelatter can tear FG-nup segments readily away to form a wider space for passage.To further examine the proposed mechanism through simulations, one transportreceptor protein, NTF2, was then embedded into one final brush-like structure andits interaction with the brush was investigated. Although constrained by the brushbundles, multiple amino acid pairs of phenylalanine and glycine could indeed bindto the transport receptor protein NTF2 very quickly. This observation further confirmsthat the bundle-based brush-like structure does offer a favorable environmentfor transport receptor proteins. In the next year, the simulations will focus on invitro NPC models engineered by our collaborators.BIBLIOGRAPHY[1] B. Fahrenkrog, J. Koser, and U. Aebi. The nuclear pore complex: A jack of alltrades? Trends Biochem. Sci., 29:175?182, 2004.[2] B. Fahrenkrog and U. Aebi. The nuclear pore complex: Nucleocytoplasmic transportand beyond. Nat. Rev. Mol. Cell Biol., 4:757?766, 2003.[3] S. A. Adam. The nuclear pore complex. Gen. Biol., 2:reviews0007.1?0007.6, 2001.[4] I. G. Macara. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev.,65:570?594, 2001.[5] M. P. Rout and J. D. Aitchison. The nuclear pore complex as a transport machine.J. Biol. Chem., 276:16593?16596, 2001.[6] D. Gorlich and U. Kutay. Transport between the cell nucleus and the cytoplasm.Annu. Rev. Cell. Dev. Biol., 15:607?660, 1999.[7] S. M. Liu and M. Stewart. Structural basis for the high-affinity binding of nucleoporinNup1p to the Saccharomyces cerevisiae importin-beta homologue, Kap95p.J. Mol. Biol., 349:515?525, 2005.[8] J. Morrison, J. Yang, M. Stewart, and D. Neuhaus. Solution NMR study of theinteraction between NTF2 and nucleoporin FxFG repeats. J. Mol. Biol., 333:587?603, 2003.[9] J. Bednenko, G. Cingolani, and L. Gerace. Importin-beta contains a COOH-terminalnucleoporin binding region important for nuclear transport. J. Cell Biol., 162:391?401, 2003.[10] R. Bayliss, T. Littlewood, L. A. Strawn, S. R. Wente, and M. Stewart. GLFGand FxFG nucleoporins bind to overlapping sites on importin-beta. J. Biol. Chem.,277:50597?50606, 2002.[11] T. A. Isgro and K. Schulten. Binding dynamics of isolated nucleoporin repeat regionsto importin-beta. Structure, 13:1869?1879, 2005.[12] T. A. Isgro and K. Schulten. Association of nuclear pore FG-repeat domains toNTF2 import and export complexes. J. Mol. Biol., 366:330?345, 2007.[13] T. A. Isgro and K. Schulten. Cse1p binding dynamics reveal a novel binding patternfor FG-repeat nucleoporins on transport receptors. Structure, 15:977?991, 2007.[14] L. Miao and K. Schulten. Transport-related structures and processes of the nuclearpore complex studied through molecular dynamics. Structure, 17:449?459, 2009.NIHMSID: NIHMS102729.[15] A. Y. Shih, A. Arkhipov, P. L. Freddolino, and K. Schulten. Coarse grained proteinlipidmodel with application to lipoprotein particles. J. Phys. Chem. B, 110:3674?3684, 2006.[16] S. J. Marrink, A. H. de Vries, and A. E. Mark. Coarse grained model for semiquantitativelipid simulations. J. Phys. Chem. B, 108:750?760, 2004.[17] W. Humphrey, A. Dalke, and K. Schulten. VMD ?Visual Molecular Dynamics.J. Mol. Graphics, 14:33?38, 1996.[18] J. C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot,R. D. Skeel, L. Kale, and K. Schulten. Scalable molecular dynamics with NAMD.J. Comp. Chem., 26:1781?1802, 2005.
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