Structure of the exportin Xpo4 in complex with RanGTP and the hypusine-containing translation factor eIF5A

Abstract

Xpo4 is a bidirectional nuclear transport receptor that mediates nuclear export of eIF5A and Smad3 as well as import of Sox2 and SRY. How Xpo4 recognizes such a variety of cargoes is as yet unknown. Here we present the crystal structure of the RanGTP·Xpo4·eIF5A export complex at 3.2 Å resolution. Xpo4 has a similar structure as CRM1, but the NES-binding site is occluded, and a new interaction site evolved that recognizes both globular domains of eIF5A. eIF5A contains hypusine, a unique amino acid with two positive charges, which is essential for cell viability and eIF5A function in translation. The hypusine docks into a deep, acidic pocket of Xpo4 and is thus a critical element of eIF5A's complex export signature. This further suggests that Xpo4 recognizes other cargoes differently, and illustrates how Xpo4 suppresses – in a chaperone-like manner – undesired interactions of eIF5A inside nuclei.

Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation

“…The hypusine-modified lysine in the GAB primary structure (K-336) is located in an unfolded segment of the protein, a short turn of 9 residues connecting two alpha helices in a sequence highly enriched in polar amino acids (KKCFPKGVD), which strongly suggest that this hypusinated lysine is exposed to the external medium. Interestingly, the exposed hypusine of eIF5A becomes essential for interaction with exportin Xpo4 allowing its nucleocytoplasmic shuttling 59 . Thus, the exposed hypusine residue might be a structural determinant needed for nuclear import of GLS2 through nuclear transport receptors or for specific interaction with nucleotides required in a putative transcriptional regulatory role, although further experiments will be needed to assess the functional relevance of this PTM in the GLS2 protein expressed in human cancer cells, as well as the role of the hypusination enzyme machinery in cancer growth and proliferation.…”

Hypusine, a polyamine-derived amino acid critical for eukaryotic translation

“…The crystal structure of the eIF5A-yeast 80S ribosome complex suggests a possible connection between eIF5A-ribosome association and the conformational changes of the ribosome during protein synthesis (90). The hypusination of eIF5A not only confers its activity in translation elongation and termination, but also dictates its subcellular localization in the cytoplasm (91), the proper compartment for protein synthesis, with the aid of the nuclear export factor, exportin 4, which recognizes the hypusinated form of eIF5A (92).…”

Structural Basis for Selective Binding of Export Cargoes by Exportin-5

“…Although this helical arrangement of HEAT20 might contribute to the compact structure of Exp-5 alone, HEAT20 is disordered in the complex structures and may not be involved in pre-miRNA or RanGTP binding from the X-ray structures. Such arrangement of Exp-5's HEAT20 resembles HEAT20 of Xpo4 alone, which interacts with the loop of HEATs 2-3 of Xpo4 alone to stabilize the toroid structure of the export complex (Aksu et al, 2016). Cse1p alone and CRM1 alone structures also showed closed conformation, which might be more stable than open conformation (Cook et al, 2005;Saito and Matsuura, 2013).…”

“…Cargo selection by conformational change of exportin on RanGTP binding is an interesting control mechanism. Since the report on the X-ray structure of RanGTP complexed with Cse1p (exportin 2) and importin-a by Matsuura and Stewart (2004), several RanGTP-related nuclear export complex structures reflecting their nuclear states have been determined by X-ray crystallography, including Exp-5:RanGTP:pre-miRNA (Okada et al, 2009), Exp-t:RanGTP:tRNA , CRM1-related complexes (Dong et al, 2009;Koyama and Matsuura, 2010;Monecke et al, 2009), and Xpo4:RanGTP:eIF5A complex structure (Aksu et al, 2016). Although recent CRM1-related structures have shown a detailed regulation mechanism of export complex, such mechanism is not generally applicable to other exportins such as Cse1p, Exp-t, and Exp-5 because these exportins or importins generally bind cargoes using the inner surface of their tandem HEAT, whereas CRM1 has a leucine-rich nuclear export signal binding surface on the outer convex surface of CRM1.…”