Correlation of CRM1-NES affinity with nuclear export activity

Abstract

CRM1 (Exportin1/XPO1) exports hundreds of broadly functioning protein cargoes out of the cell nucleus by binding to their classical nuclear export signals (NESs). The 8- to 15-amino-acid-long NESs contain four to five hydrophobic residues and are highly diverse in both sequence and CRM1-bound structure. Here we examine the relationship between nuclear export activities of 24 different NES peptides in cells and their CRM1-NES affinities. We found that binding affinity and nuclear export activity are linearly correlated for NESs with dissociation constants ( K_ds) between tens of nanomolar to tens of micromolar. NESs with K_ds outside this range have significantly reduced nuclear export activities. These include two unusually tight-binding peptides, one from the nonstructural protein 2 of murine minute virus (MVM NS2) and the other a mutant of the protein kinase A inhibitor (PKI) NES. The crystal structure of CRM1-bound MVM NS2^NES suggests that extraordinarily tight CRM1 binding arises from intramolecular contacts within the NES that likely stabilizes the CRM1-bound conformation in free peptides. This mechanistic understanding led to the design of two novel peptide inhibitors that bind CRM1 with picomolar affinity.

FIGURE 1:

Correlation of NES export activity with binding affinity to CRM1. (A) Schematic of the workflow to quantify the ratio of cytoplasmic to nuclear mean fluorescence intensity (R_C/N). R_C/N values are used as a measurement of NES activity in living HeLa cells. The loop in the workflow indicates that this process is repeated for at least 30 representative cells collected from at least three independent experiments (listed in Table 2) for statistical analysis. (B) Leptomycin B (LMB) sensitive nuclear export activity of EYFP₂-SV40^NLS-NES fusion proteins in HeLa cells. YFP (pseudocolored in yellow) and Hoechst (pseudocolored in blue) images were captured using spinning disk confocal microscope (40×). From left to right, PKI^NES, HIV Rev^NES, and CDC7^NES. Nuclear accumulation after treatment with 5 nM leptomycin B (+LMB) for 16–18 h demonstrates CRM1-dependent export. Representative images of all other NESs are shown in Supplemental Figure S1A. (C) Correlation of in vitro affinity and in vivo nuclear export activity. In vitro binding affinities of 24 NESs (includes two negative controls PKI^NES(I47A) and PKI^NES(L42A/L45A)) are plotted as a function of their nuclear export activities. Error bars represent 95% confidence intervals. Half of the vertical error bars for the binding affinities of PKI^NES(L42A/L45A), Super PKI^NES, and MVM NS2^NES are missing because the upper or lower limits are undetermined. The Pearson’s r value of the 24 NESs is –0.65 (p = 0.0006) and is –0.87 (p = 1.48 × 10^-7) when Super PKI^NES and MVM NS2^NES are not included.

FIGURE 2:

Inhibitory effect of extraordinary tight-binding NESs. (A) EYFP₂-SV40^NLS-PKI^NES is cotransfected with RFP, RFP-MVM NS2^NES, RFP-Super PKI^NES, RFP-PKI^NES, or RFP- X11L2^NES and compared with cells expressing EYFP₂-SV40^NLS-PKI^NES that are treated with small molecule inhibitor LMB. (B) EYFP₂-SV40^NLS-X11L2^NES is cotransfected with RFP, RFP-MVM NS2^NES, RFP-Super PKI^NES, RFP-PKI^NES, or RFP-X11L2^NES and compared with cells expressing EYFP₂-SV40^NLS-PKI^NES that are treated with small molecule inhibitor LMB. The numbers of examined cells from at least three independent experiments are indicated in parentheses in A and B. Error bars represent SD. The p values were calculated in comparison to control (reporter only) using Mann–Whitney tests. Note that observed R_C/N values are presented without normalization to compare with R_C/N on LMB treatment (+LMB). (C) Representative images of cells transfected with EYFP₂-SV40^NLS-PKI^NES (reporter) and RFP-tagged NESs (competitive peptides). YFP (pseudocolored in yellow), Hoechst (pseudocolored in blue), and RFP (pseudocolored in red) images were captured using spinning disk confocal microscope (40×). The expression levels of reporter proteins and competitive peptides and are summarized in Supplemental Figure S1C.

FIGURE 3:

Crystal structure of MVM NS2^NES bound to CRM1 and design of picomolar affinity peptide inhibitors. (A) Left, MVM NS2^NES (pink cartoon) binds CRM1 (gray surface) like a typical class 1a NESs, with an N-terminal helix and C-terminal strand. Right, Details of CRM1-NES interaction. Black dotted lines show polar contacts within the NES (pink cartoon) and between the NES and CRM1 (gray cartoon). (B) Final refined model of the NES (pink sticks) overlaid onto electron density meshes of 2mFo-DFc map contoured at 1.0σ and kick OMIT map (calculated by omitting the NES peptide) contoured at 3.0σ. (C) Sequences of wild-type NES sequences and their chimera with MVM NS2^NES. Φ side chains in the NESs that bind hydrophobic pockets in the CRM1 groove are numbered and underlined. (D) Differential bleaching data of MBP-Super PKI-NS2^NES and MBP-CDC7-NS2^NES binding to CRM1. Direct titrations of FITC-PKI^NES to CRM1 and RanGTP are shown in black and competition titrations of MBP-NESs to FITC-PKI^NES, CRM1, and RanGTP are shown in color. Dissociation constants (K_d) are obtained from triplicate titrations and 95% confidence intervals are reported in brackets. U = cannot be defined.