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Cp-NTF2: Cryptosporidium parvum Nuclear Transport Factor 2

PDB entry: 1ZO2

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Cryptosporidium parvum is a protozoan parasite from the phylum Apicomplexa, which also includes Plasmodium species and Toxoplasma gondii. It is the causative agent for cryptosporidiosis in humans and animals, and may be transmitted via contaminated water and food, as well as contact with infected animals and humans.

Typical symptoms are cramps, fever, diarrhea and weight loss, but may be more serious and particularly persistent in patients with compromised immune systems, such as those with AIDS or cancer.

We have solved the structure of nuclear transport factor 2 protein (NTF2) from C. parvum, namely cgd3_300, an ortholog of PF14_0122, the Plasmodium falciparum NTF2, with 42% sequence identity between the two.

NTF2 is a 14 kDa homodimeric protein that is involved in the transport of marcromolecules between the cell nucleus and the cytoplasm. Nuclear pore complexes (NPCs) are the large macromolecular assembles (comprised of ~30 proteins for yeast and 30-50 proteins for vertebrates and are collectively termed nucleoporins) that span the nuclear envelope and mediate the transport of a variety of macromolecules. Nucleoporins contain large repeats of FG (often FxFG or GLFG) that comprise the hydrophobic Phe-rich core that is separated by hydrophilic linker regions. Although the precise mechanism of macromolecular cargo transport across the nuclear envelope is not well defined, there is strong evidence supporting the direct interaction between the carrier protein and the FG nucleoporins.

The passage of macromolecules through the NPCs is mediated by carrier proteins like NTF2 that shuttle proteins between the cytoplasm and cell nucleus by binding their cargo protein in one compartment and releasing it in the other.1 For example, the classical nuclear localization sequence (NLS) is mediated by importin-beta which binds its cargo in the cytoplasm (via the importin-alpha adapter). This complex is then translocated across the NPC; and in the nucleus RanGTP binds to the importin-beta complex to dissociate the complex, releasing the cargo. Importin-beta is returned to the cytoplasm in complex with the associated RanGTP. Once in the cytoplasm, RanGAP catalyzes the hydrolysis of the GTP bound to Ran and facilitating the dissociation of the Ran-importin-beta complex, so the process can begin again. This cycle leads to a depletion of RanGTP in the nucleus and an excess of RanGDP in the cytoplasm and necessitates that RanGDP be imported to the nucleus where it can be converted to RanGTP which is mediated by the nuclear Ran guanine nucleotide exchange factor (RanGEF), RCC1. NTF2 is responsible for the translocation of RanGDP.2

The overall structure of Cp-NTF2 is similar to those crystallized for Saccharomyces cerevisiae (yNTF2) and Rattus norvegicus (rNTF2). There is 40-50% sequence identity between Cp-NTF2, yNTF2, rNTF2 and PF14_0122. The Kd for the interaction of NTF2 with RanGDP has been determined to be 100 nM3 and for the interaction of NTF2 with FxFG repeat peptide at 1 ÁM.4 The Kd for dimerizationi is 1.1 ÁM which is significant in vivo, where the concentration of NTF2 in the nuclear envelope is 20 ÁM, as compared to the cytoplasm and nucleoplasm at 0.3 ÁM.5 Site direct mutagenesis and crystal structures have facilitated the study of this biologically important dimerization interaction that may constitute an additional form for regulation of the protein binding to RanGDP and FxFG nucleoporin.

There are structures available for both rNTF2 and yNTF2 for with FxFG nucleoporins.6,7 A hydrophobic patch centred around Trp7 in rNTF2 that forms an essential interaction with the hydrophobic FxFG nucleoporins aligns with Phe10 in CP-NTF2, also in a hydrophobic patch with similarity to rNTF2. The importance of Trp7 in rNTF2 has been established with a series of experiments including site-directed mutagenesis, protein-protein interaction studies, and functional assays conducted in vitro and in vivo. Although a key interaction has been found, there are many questions that remain concerning this interaction, as other factors must be active in order to explain the experimental results. Further studies on the crystal structure of yNTF2-N77Y complexed with FxFG peptide have further defined the interaction. The FxFG binding site on yNTF2 contains residues from both chains of the dimeric molecule. The interaction involves a change in the conformation of the side chains in order to incapsulate the Phe rings the peptide.

There are structures available for the RanGDP complex with rNTF2.8 The crystal structure indicates that 3 equivalents of Ran interact with the homodimer rNTF2. Two equivalents interact in a similar manner over a significant interface, whereas the third equivalent interacts with the two rNTF2 chains in a different orientation with a small interface. The interaction between the interface of rNTF2 involves a hydrophobic interaction of the Ran Phe72 and conserved hydrophobic groups of NTF2 (for example, rNTF2 Phe119 (and likely correspondingly CP-NTF2 Phe122) and rNTF2 Leu89, Lys90, and Ala91 (and CP-NTF2 Val92, Arg93, and Ile94, respectively)).

References

1 R. Bayliss, A. H. Corbett, M. Stewart (2000), Traffic, 1, 448-456.
2 M. Stewart (2000), Cell Struct. Funct., 13025, 217-225.
3 R. Bayliss, K. Ribbeck, D. Akin, H. M. Kent, C. M. Feldherr, D. Gorlich, M. Stewart (1999), J. Mol. Biol, 293, 579-593.
4 C. Chaillan-Huntington, C. Villa-Braslavsky, C. Kulmann, M. Stewart (2000) J. Biol. Chem., 275, 5874-5879.
5 C. Chaillan-Huntington, P. J. G. Butler, J A. Huntington, D. Akin, C. Feldherr, M. Stewart (2001), J. Mol. Biol., 314, 465-477.
6 R. Bayliss, S. W. Leung, R. P. Baker, B. B. Quimby, A. H. Corbett, M. Stewart (2002) The EMBO Journal, 21, 2843-2853.
7 I. Cushman, B. R. Bowman, M. E. Sowa, O. Lichtarge, F. A. Quicho, M. S. Moore (2004) J. Mol. Biol., 344, 303-310.
8 M. Stewart, H. M. Kent, A. J. McCoy (1998) J. Mol. Biol., 277, 635-646.

Materials and Methods