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The Nuclear Pore Complex

Gateway to the Nucleus

The nucleus of the cell is centrally important to an organism. It serves to store and organize genetic information, the atomic blueprint for the organism, while separating and protecting this very important information from the host of other cellular components. While the nucleus requires this protective isolation, it also needs to communicate with the rest of the cell, exchanging proteins and RNA, for a variety of nuclear and cytoplasmic processes which act in concert. The nuclear pore complex (NPC), perhaps the largest protein complex in the cell, is responsible for the protected exchange of components between the nucleus and cytoplasm and for preventing the transport of material not destined to cross the nuclear envelope.

Much is known about the behemoth complex from a variety of experimental techniques, but its vast size has made pointed study quite difficult. The NPC has a mass of ~125 megaDaltons (MDa) in vertebrates () and 66 MDa in yeast (). It has an octagonally symmetric cylindrical structure around the axis of transport and a planar pseudo-symmetry through the nuclear envelope. The yeast NPC has been well characterized and is thought to contain an upper limit of 30 distinct types of proteins (termed nucleoporins, or nups) (). The number is low and rather surprising considering that the ribosome, another macromolecular protein complex, contains 75 different types of proteins and weighs only 4 MDa. The body of the vertebrate NPC is approximately 145 nm in diameter and 80 nm length across the nuclear envelope. The yeast NPC is smaller at approximately 96 nm in diameter and 35 nm in length (). The NPC is truly large, especially when compared to other proteins or protein complexes in the cell. For comparison, a tetramer of aquaporin proteins measures only 6.5 nm across!

Nucleocytoplasmic Transport

The immense size of the NPC makes it difficult to study the system in detail both experimentally and computationally. However, a variety of experiments have elucidated many properties of the NPC and the transport process itself. They have described the macroscopic structure of the NPC as well as other features which provide the foundation for computational efforts to tackle the mechanism of nucleocytoplasmic transport.

Transport Cycle. In order to pass through the NPC, a large molecule (cargo) must associate with another protein called a transport receptor. These transport receptors then act as chaperones which shuttle cargo through the NPC. Recognition by the transport receptor takes place via a specific sequence of amino acids in the cargo protein. Besides cargo and transport receptors, the third ingredient necessary for nuclear transport is the signaling protein Ran, which hydrolyzes GTP. Ran is responsible for regulating the interaction of transport receptor and cargo (, ), and RanGDP/RanGTP concentration gradients across the nuclear envelope drive nuclear import and export. Once a transport receptor identifies and binds its cargo, the transport complex may be imported or exported through the NPC. The mechanism by which the NPC selectively allows the transit of import or export complexes, while restricting the passage of inert species is poorly understood. However, nups with repeating amino acid sequences involving phenyalanine (F) and glycine (G) have been thoroughly implicated in the process.

FG-Nups. The NPC requires the presence of nups with FG sequence repeats. Many transport receptors, such as importin-β (, , ) and NTF2 (, , , ), have been shown to have hydrophobic binding sites on their surface for these "FG-Nups". Furthermore, the systematic deletion of the FG-repeat regions from FG-Nups in yeast NPCs has been shown to be lethal to yeast cells (). The deletion of the FG-repeat region from one nup, nup116p, caused cell death on it own. FG-Nups have also been shown to form filaments () and colocalize with the filaments on both sides of the NPC. This evidence is consistent with the FG-Nups forming the filaments of the NPC, to which transport complexes may dock in the initial and final stages of transport. Finally, the FG-Nups have been shown to be highly disordered and contain almost no secondary structure (). Localization of nups in the yeast NPC () shows that many of the FG-Nups occupy the central pore region of the NPC, through which the transport complexes pass. All this evidence points toward intimate interactions which must take place between FG-Nups and transport receptors to make transport successful.

Discovering New Transport Receptor Interactions

In order to understand the mechanism of nucleocytoplasmic transport, one must understand the manner in which the NPC interacts with transport receptors. To examine these interactions, we have performed simulations of transport receptors in a solution containing short chains of FG-Nups. We have examined both the transport receptors importin-β and NTF2. During the course of any single simulation, FG-Nups were found to diffuse through the solution and bind to the surface of the transport receptor. Some of these interactions are likely to play a role in the in vivo recognition of the transport receptor by the NPC, and allowing it passage into or out of the nucleus.

To determine which of the several binding events occurring in a simulation are relevant, we also aligned several sequences of the transport receptor, with each set of aligned residues receiving a score based on conservation, evolutionary distance (see our tutorial on sequence alignment [pdf]), and hydrophobicity. Highly scoring residues were regarded as "hot spots" for the binding of FG-Nups on the surface of the transport receptor (see importin-β figure on the right).

Importin-β. We simulated a crystal structure () of the transport receptor importin-β with cargo, fully solvated and ionized, in the presence of FG-Nups, and performed a sequence alignment using eight importin-β species. Using this combination of molecular dynamics simulations and sequence alignment, we were able to confirm the existence of 3 out of 4 binding spots which were known previously from experiments (, , ). Furthermore, we were able to identify six novel binding spots for FG-Nups which were previously unknown. (One of these spots was identified independently by experimentalists ().) The simulations provide the first atomic-scale look at these novel binding spots for FG-Nups, and suggest that the extent of binding on the surface of importin-β is much larger than previously realized. The additional binding spots on the surface of importin-β suggest that the molecule has a more extensive ability to interact with the NPC than previously expected and also implies that other transport receptors similar in size harbor an equally large number of binding spots.

NTF2. The transport receptor NTF2 is a vital in maintaining the proper balance of components across the nuclear envelope to drive nuclear import and export. The protein Ran is an enzyme which catalyzes the hydrolysis of GTP. The energy released upon hydrolysis is used to disassemble transport complexes which have been exported to the cytoplasm. Alternatively, the binding of RanGTP to transport receptors may initiate transport complex formation (for those to be exported) or initiate disassembly (for those which have been imported). Maintaining the proper balance of Ran across the nuclear envelope is crucial for nuclear transport. The reimporting of RanGDP to the nucleus, where it is charged to become RanGTP, is the job of NTF2.

We performed two sets of simulations on NTF2. One set involved NTF2 with RanGDP bound (), as the transport complex would appear upon import into the nucleus. The other set involved the simulation of NTF2 alone (), as the dimer would appear upon export. We simulated both complexes in a solution of short chains of FG-Nups for a total of over 250 ns to determine how and where the chains interacted with the NTF2 surface, and aligned the sequences of 15 distinct NTF2 species to support the simulation findings. Using the technique, we were able to verify the existence of four binding spots on the NTF2 surface already discovered via previous X-ray (), mutational (, , ), NMR (), and computational data (). We also discovered two novel binding spots, not yet discovered by experiment. All six binding spots broadly form a stripe across the surface of NTF2, as indicated in the figure below. The regularity and proximity of binding spots play a role in the identification of the NTF2 by NPC FG-Nups. Furthermore, the novel binding spots appear on a structurally variable part of the NTF2 surface, suggesting that these spots, along with the Ran binding spot, may play a role in tuning the transport of NTF2 for import or export.

The existence of numerous binding spots on the surface of any transport receptor can serve to distinguish it from other proteins which reside around the nuclear envelope or it may enable the NPC to more finely tune the transport of cargo. Upon examination of a third transport receptor, Cse1p, the case becomes convincing for numerous binding spots on the transport receptor surface as a necessity for nuclear transport.

Cse1p. Cse1p is a yeast transport receptor which functions as the Kap60p exporter. (Cse1p is known as CAS in vertebrates and Kap60p is importin-α) (, , , ). The transport receptor recycles Kap60p back to the cytoplasm after it assists in nuclear import as an adaptor for Kap95p (importin-β). The crystal structure of Cse1p is available in complex with Kap60p and RanGTP () and in cargo-free form (). Like importin-β, Cse1p is a right-handed superhelical protein. It is composed of 20 HEAT repeats. Kap60p is comprised primarily of 10 Armadillo (ARM) repeats.

We performed four molecular dynamics simulations on the Cse1p:Kap60p:RanGTP export complex, similar to those performed on importin-β and NTF2, to study interactions between FG-nup peptides and the surface of the transport complex. The simulations revealed 14 binding spots for FG-repeat peptides on the surface of Cse1p and five binding spots on the surface of Kap60p. They are particularly valuable in this case because little study has been devoted to detailing the interactions of FG-Nups with Cse1p compared to the large body of evidence uncovered for importin-β and NTF2. We found binding of FG-Nups to be concentrated between HEAT repeats 10-16, with eight of the 14 binding spots appearing in that region and one or more binding spots for each crevice between repeats. Binding of FG-nup peptides to Kap60p, on the other hand, appears to be much less extensive and sparser than on the Cse1p surface at only five spots on the Kap60p surface, indicating a much lesser concentration in binding spots than seen on the Cse1p surface. The similarities in FG-Nup binding between Cse1p and the transport receptors importin-β and NTF2 and the differences in binding of Kap60p, an inert macromolecule that cannot traverse the nuclear pore alone, provide a unique insight into how the nuclear pore likely recognizes a transport receptor (and cargo) destined for nuclear import, while preventing the transport of other molecules.

Comparing the binding spots on the surfaces of the three transport receptors with that of Kap60p, reveals a striking difference, not simply in the number of binding spots, but most notably in their orientation on the surfaces of each protein. The relatively small amount of Kap60p binding spots yields a lower average binding spot density across the entire protein surface than across that of Cse1p, for example. Furthermore, the Kap60p binding spots are also spread widely across the protein's surface from ARM repeats 1-9, with no more than two binding spots clustered at short distances. The location of Cse1p binding spots, on the other hand, appears in three broader clusters across the surface, the most populated one near the beginning of the C-terminal half of the molecule: eight of the 14 Cse1p binding spots are located in this cluster between HEAT repeats 10-16. The binding spots in this cluster are separated from their neighboring binding spots by regular distances of ~16 Å (± 4 Å). Importin-β and NTF2 exhibit similar sets of concentrated binding spots on their surfaces.

The multitude of binding spots separated by regular and short distances on the surfaces of the Cse1p, importin-β, and NTF2 compared to that of Kap60p suggests that the NPC uses both binding spot number and proximity to determine whether a macromolecule (complex) is fit for transport. In distinguishing between legitimate transport receptors and inert molecules not destined to traverse the nuclear envelope alone, the NPC must utilize a mechanism which is capable of protecting against the random occurrence of a binding spot on the surface of an inert molecule that is capable of binding FG-nups. Indeed, any inert macromolecule may possess one or several hydrophobic depressions on its surface not explicitly for nuclear transport but which are capable of FG-nup binding nonetheless. The binding of a single FG-repeat, or even a small few, to the surface of a molecule is unlikely to render successful nuclear transport. The NPC may protect against these "random" binding spots by requiring a transport receptor to have several binding spots in close proximity on its surface. With numerous and close binding spots on a transport receptor's surface, the NPC may be able to bind multiple FG-repeats to the transport receptor simultaneously, ensuring a strong enough composite binding energy to enable nuclear transport. Furthermore, the regular spacing between binding spots may be coordinated with the regular spacing between FG- repeats within individual FG-nups. Both spacings may be tuned to allow the binding of FG-repeats to binding spots, while also allowing the energetically favorable adherence of FG-nup linker regions to the transport receptor surface. This regular and numerous binding of FG-nups may effectively serve as a robust means for the NPC to identify transport complexes among a host of inert molecules that may possess hydrophobic patches on their surfaces.

Publications and News

• Cse1p binding dynamics reveal a novel binding pattern for FG-repeat nucleoporins on transport receptors. Timothy A. Isgro and Klaus Schulten. Structure, 15:977-991, 2007.

• Association of nuclear pore FG-repeat domains to NTF2 import and export complexes. Timothy A. Isgro and Klaus Schulten. Journal of Molecular Biology, 366:330-345, 2007.

• Binding dynamics of isolated nucleoporin repeat regions to importin-b. Timothy A. Isgro and Klaus Schulten. Structure, 13:1869-1879, 2005.

• See a review of our recent work in the journal Structure.

• See another review of our earlier work in Structure, as well as a review in the journal Science in their feature Editors' Choice.

• Our work was featured as a success story for computing at both the Pittsburgh Supercomputing Center and the National Center for Supercomputing Applications.

• Our work was also featured at WhyFiles.org, a website dedicated to accessible explanations of scientific research for the novice scientist and layperson.

Investigators

• Tim Isgro
• Yi Wang
• Klaus Schulten
• Murray Stewart (University of Cambridge)
• Andrej Sali (UCSF)
• Mike Rout (The Rockefeller University)
• Reiner Peters (Münster University)

Page created and maintained by Tim Isgro.

Beckman Institute for Advanced Science and Technology // National Institutes of Health // National Science Foundation // Physics, Computer Science, and Biophysics at UIUC
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