Electrostatic Interactions Involving the Extreme C Terminus of Nuclear Export Factor CRM1 Modulate Its Affinity for Cargo

Abigail M Fox,Danguole Ciziene,Stephen H Mclaughlin,Murray Stewart

doi:10.1074/jbc.m111.245092

Abigail M Fox, Danguole Ciziene + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.245092

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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.

Highlights

Nuclear protein export is a fundamental function in all eukaryotes and is crucial for maintaining the appropriate nucleocytoplasmic distribution of a broad range of transcription factors, signaling molecules, and cell cycle regulators, as well as viral components such as the HIV Rev protein and the influenza NS2 protein
Function of CRM1 C-terminal Helix in nuclear export signal (NES) Binding than in the cytoplasm when Ran has been removed. This change in affinity could be due to changes in the positions of the A-helices of HEAT repeats 11 and 12 that result from a contribution of local changes induced via an autoinhibitory mechanism of the HEAT 9 loop proposed by Koyama and Matsuura (11) and/or a larger, global change in CRM1 conformation as proposed by Dong et al (13) and Monecke et al (10)
We propose a model in which the C-terminal helix and the HEAT loop 9 induce local conformational changes that stabilize the closed conformation of the CRM1 NES binding site, which contributes to the release of NES-containing cargoes into the cytoplasm following the removal of Ran and to preventing the return of cargoes to the nucleus

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—S. cerevisiae Xpo1p was expressed from the pET30TB plasmid (a derivative of pET30aTEV in which the BamHI site in the TEV sequence has been removed) that adds a His6-S tag followed by a TEV cleavage site to the N terminus (14). Clarified lysate containing soluble His6-tagged protein was mixed with the NiNTA resin and incubated at room temperature for 45 min. The resin was centrifuged at 1,500 rpm for 5 min at 4 °C and washed three times with 50 ml of binding buffer. Either clarified lysate (ϳ0.5 ml) or purified protein was added to the resin and incubated at room temperature for 45 min. Beads were sedimented by centrifugation at 2,000 rpm for 3 min in a microcentrifuge and washed three times with 1 ml of binding buffer to remove excess bait protein. The binding of increasing concentrations of wild type and mutant CRM1 protein to 10 –50 nM YFP-PKI-NES fusion protein (11) was measured by fluorescence anisotropy at 25 °C using a PerkinElmer LS55 fluorimeter equipped with a Hamilton Microlab titrator controlled by laboratory software. The fraction of YFP-NES bound at each concentration of titratant, fb, was calculated using the fitted constants from the equation above using the following

F Ϫ F0 F1 Ϫ F0

RESULTS

DISCUSSION