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    • The small GTPase Ran regulates karyopherin

    2021-05-13

    The small GTPase Ran regulates karyopherin–cargo interactions and the directionality of karyopherin-mediated transport. Like other Ras-family GTPases, Ran cycles between GTP- and GDP-bound states, and the two surface loops in Ran, referred to as the switch I and switch II loops, undergo significant conformational changes between the GTP- and GDP-bound states. In addition, Ran has a C-terminal extension that is disordered in the GTP-bound state but folds back against the body of Ran as an α-helix in the GDP-bound state. The conformations of these three regions (switch I, switch II, and the C-terminal extension) that are sensitive to the nucleotide state of Ran are important in determining its interactions with other proteins. The low intrinsic rates of nucleotide exchange and hydrolysis on Ran are stimulated by specific factors in vivo. Ran GTPase activity is stimulated by the order of 105 by RanGAP, whereas nucleotide exchange is stimulated by the Ran guanine nucleotide exchange factor, RCC1. Based on the localizations of RanGAP in the FAK Inhibitor 14 synthesis and RCC1 in the nucleoplasm, cytoplasmic Ran is primarily in the GDP-bound state whereas nucleoplasmic Ran is kept primarily in the GTP-bound state. This gradient of Ran nucleotide state is an important determinant of the directionality of nuclear transport. In nuclear import, RanGTP competes with the cargoes to bind to importins, allowing the cargo binding in the cytoplasm and RanGTP-mediated cargo dissociation in the nucleus. By contrast, in nuclear export, RanGTP and cargoes bind cooperatively to exportins in the nucleus, and the exportin–RanGTP–cargo complexes are disassembled in the cytoplasm, where RanGTPase is activated by RanGAP and the Ran-binding proteins RanBP1/2. Thus, the association and dissociation of karyopherin–cargo complexes are regulated by direct binding of Ran in a compartment-specific manner. CRM1 (also known as exportin 1 or Xpo1) is the most versatile exportin that facilitates nuclear export of a broad range of cargoes.[8], [9], [10], [11] The majority of the export substrates of CRM1 contain a short peptide sequence (10–15 residues), the so-called leucine-rich nuclear export signal (NES) that was first identified in cAMP-dependent protein kinase inhibitor (PKI) and HIV-1 Rev. Leucine-rich NESs typically harbor four or five characteristically spaced hydrophobic residues that are crucial for the binding to CRM1. Although the NESs show considerable sequence diversity,[14], [15] recent structure determination of CRM1–cargo complexes with and without RanGTP[16], [17], [18] showed that at least three NESs (leucine-rich NES of snurportin, PKI, and Rev) share the ability to bind specifically to the same site: a hydrophobic cleft of CRM1. CRM1 is a ring-shaped molecule that is constructed from 21 tandem HEAT repeats, each of which consists of two antiparallel α-helices, designated A-helix and B-helix, connected by loops of varying length. The A-helices form outer convex surface whereas the B-helices form the inner concave surface. The hydrophobic cleft on the convex outer surface of CRM1, formed between the A-helices of HEAT repeats 11 and 12, constitutes the NES-binding site. The hydrophobic side chains of NESs fit into five hydrophobic pockets along this cleft. The binding site of Leptomycin B, a potent inhibitor of CRM1-mediated nuclear export, is located in this hydrophobic cleft, and so it is likely that this cleft is the general binding site for NESs. CRM1 is unusual among karyopherins in that it has a cargo-binding site on its outer surface (instead of its inner surface),[4], [20], [21], [22], [23] but this is important for CRM1 to carry a broad range of cargoes that vary greatly in size and shape, including huge cargoes such as ribosomal subunits. In contrast to the cargoes that bind to the outer surface of CRM1, RanGTP binds to the inner surface of CRM1.[17], [18] Four distinct regions of CRM1 contribute to RanGTP binding, and CRM1 directly binds to both switch I and switch II loops of RanGTP, accounting for the ability of CRM1 to discriminate between GTP- and GDP-bound Ran. Interestingly, the C-terminal α-helix of CRM1 (the C-helix) adopts dramatically different positions depending on whether or not RanGTP is bound to the CRM1–cargo complexes.[16], [17] In the binary CRM1–snurportin complex, the C-helix lies across the central cavity of the CRM1 ring with its C-terminus located close to the NES-binding site, whereas in the ternary CRM1–snurportin–RanGTP complex, the C-helix is located on the outer surface of the CRM1 ring, indicating that the C-terminus of CRM1 could regulate the affinity of NES in a way that is sensitive to RanGTP.