title: Electrostatic interactions involving the extreme C terminus of nuclear export factor CRM1 modulate its affinity for cargo.
abstract: The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A) α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Abigail M
authors.initials: AM
authors.lastname: Fox
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Danguole
authors.initials: D
authors.lastname: Ciziene
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Stephen H
authors.initials: SH
authors.lastname: McLaughlin
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom. Electronic address: ms@mrc-lmb.cam.ac.uk.
authors.forename: Murray
authors.initials: M
authors.lastname: Stewart
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D001665:Binding Sites; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D028884:Karyopherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D000097606:Exportin 1 Protein
chemical_list: D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000097606:Exportin 1 Protein