FG-nucleoporin binding domain of Tap/NXF1

The vertebrate Tap protein is a member of the NXF family of shuttling transport receptors for nuclear export of mRNA. Tap has a modular structure, and its most C-terminal domain is important for binding to FG repeat-containing nuclear pore proteins (FG-nucleoporins) and is sufficient to mediate nuclear shuttling. We report the solution structure of this C-terminal domain, which is based on a distinctive arrangement of four -helices and is joined to the next module by a flexible 12-residue Pro-rich linker. F617A Tap suppresses FG-nucleoporin binding by the most C-terminal domain that, together with the structure of the other modules from which Tap is constructed, provides a structural context for its nuclear shuttling function. Nuclear trafficking is mediated by soluble transport factors that shuttle between the nuclear and cytoplasmic compartments through nuclear pore complexes (NPCs)1–3. Although the precise mechanism by which transport factors in complex with their cargo move through NPCs is controversial, a consensus is emerging that this step involves interactions with nuclear pore proteins containing characteristic phenylalanine-glycine (FG) repeats2–6. Vertebrate Tap (NXF1) and Saccharomyces cerevisiae Mex67p are members of the NXF family of mRNA nuclear export factors7,8. Mex67p and Caenorhabditis elegans NXF1 are essential for the nuclear export of poly(A)+ mRNA7,9. Addition of the human NXF1 homolog, Tap, to cultured human 293 cells stimulates the nuclear export of mRNAs that are otherwise exported inefficiently10, and facilitates the export of unspliced viral mRNA containing the constitutive transport element (review ref. 1). Mex67p binds Mtr2p in vitro and in vivo, and formation of this heterodimer is essential for both mRNA export and association of Mex67p with nucleoporins11. Similarly, the NTF2-like protein NXT1 is a critical cofactor in human and invertebrate cells10,12,13 and may act as a regulatory switch12,13. The ability of recombinant Tap–NXT1 heterodimers to rescue the function of yeast Mex67p/Mtr2p double knockouts implicates Tap as a general cellular factor for the export of processed mRNA14. Tap has a multidomain structure. A nuclear localization sequence and a noncanonical mRNA-binding domain followed by four LLR repeats are located in its N-terminal half15,16. The C-terminal half contains a NTF2-homology domain (residues 371–551) that forms a heterodimer with NXT1, which binds nucleoporins17, followed by a second domain that is both necessary and sufficient for localization to NPCs in vivo and nucleocytoplasmic shuttling of fusion proteins15,18. This most C-terminal domain of Tap also contains a key FG-nucleoporin binding site14,15,18–20, and sequence analysis21 suggests that it has a UBA (ubiquitin-associated) fold. We report here the solution structure of residues 551–619 of Tap. This fragment (TapC) encompasses the most C-terminal Tap domain, retains binding to FG-nucleoporins and forms a compact four-helix fold related to that of a UBA domain. The structure indicates that several previously described mutations in this region15,20,21 probably disrupt its structure rather than identifying residues directly involved in nucleoporin binding.