Supplementary Table 1. Crystallographic statistics CRM1-SNUPN complex Space group P6 4 22 a=b=250.4, c=190.4 Data collection statistics: CRM1-selenomethionine SNUPN MAD data Peak Inflection Remote Native Resolution range (Å) 50-3.0 50-2.9 50-3.1 50-2.9 Wavelength (Å) 0.97935 0.97945 0.97167 0.97934 Number of unique 69,812 69,818 63,383 76,993 reflections Mean redundancy 7.4 (7.5)* 7.4 (7.5) 7.4 (7.5) 6.1 (5.1) Completeness 99.1 (99.6) 99.1 (99.6) 99.1 (99.6) 99.5(98.2) R sym 0.085 (0.540) 0.083 (0.580) 0.093 (0.493) 0.073 (0.591) Mean I/σ 23.3 (3.7) 25.0 (4.7) 20.9 (3.9) 22.3 (2.1) Refinement (MAD inflection data): Resolution range (Å) 50.0-2.9 (2.97-2.90) Å Number of working and test reflections 69809 (4844) Number of test reflections 3667 (236) Completeness (%) 94.4 (85.4) R work /R free 0.239 (0.317) / 0.269 (0.371) r.m.s. deviation of from ideal bond length (Å) 0.011 r.m.s. deviation of from ideal bond angle (degrees) 1.47 Ramachandran analysis (%) Most favoured 89.2 Allowed region 10.1 Generally allowed region 0.7 Disallowed region 0.0 B-factors CRM1: 42.7 Å 2 SNUPN: 26.8 Å 2 *values for highest resolution shell are shown in parentheses. www.nature.com/nature 1
Supplementary Figures Figure S1. Both full length SNUPN and SNUPN(1-342) bind CRM1. In vitro pull down assays of immobilized GST-SNUPN proteins and CRM1 in the presence and absence of RanGTP. Bound proteins were visualized by SDS-PAGE and Coomassie staining. www.nature.com/nature 2
Figure S2. CRM1 binds SNUPN with higher affinity than RanGppNHp. a, ITC experiments of CRM1-SNUPN(1-342) (black) and CRM1-RanGppNHp (red) interactions. Thermodynamic parameters K D = 1.428 ± 0.002 μm, H = -13.0 ± 0.5 kcal/mol and S = -18.4 ± 1.7 kcal/mol/k for CRM1 binding to SNUPN were obtained from two independent experiments (± standard deviation). Experimental details are described in the Online Methods. b, Gel filtration chromatography of CRM1 alone (blue), a 1:1.7 molar ratio CRM1:RanGppNHp mixture (dashed magenta), Kap104p alone (yellow) and a 1:1.7 molar ratio Kap104p: RanGppNHp mixture (green). Kap104p binds RanGppNHp (K D = 30 nm; ITC data not shown) but CRM1 shows no observable binding to RanGppNHp by ITC or gel filtration. Therefore, of the CRM1-SNUPN or CRM1-RanGTP dimeric intermediates, the former is more likely to form in vitro. www.nature.com/nature 3
Figure S3. Arrangement of the CRM1 (pink) and the SNUPN LR-NES (yellow) helices at the LR-NES binding site. The SNUPN segment containing residues 1-16 (yellow) is a LR-NES. The signal binds between helices H11A and H12A of CRM1 (pink). www.nature.com/nature 4
Figure S4. Alignment of CRM1 H11A-H12A segments from 10 phylogenetically diverse eukaryotes. LR-NES contact residues that are identical to human CRM1 are shaded. 10 of the 14 CRM1 residues that contact the LR-NES are invariant and the other four are 70% identical across 10 phylogenetically diverse species. H11 and H12 are the most highly conserved CRM1 HEAT repeats (48% of their residues are invariant). www.nature.com/nature 5
Figure S5. Helical wheel schematic summarizing contacts < 4.0 Å between the LR-NES helix of SNUPN and helices H11A, H11B and H12A of CRM1. www.nature.com/nature 6
Figure S6. Sequences of LR-NESs from SNUPN, HIV1-REV, NMD3, the NS2 protein of the parvovirus Minute Virus of Mice (MVM-NS2), camp-dependent protein kinase inhibitor (PKI) and tumor suppressor protein p53. www.nature.com/nature 7
Figure S7. The LR-NES of SNUPN is a combined α-helical and extended peptide. A view of the LR-NES of SNUPN (stick drawing in yellow) bound to CRM1 (pink ribbon) focusing on the α-β transition region of SNUPN (residues 9-14). Dashed lines indicate polar contacts that are consistent with hydrogen bonds, and electron density of the simulated annealing omit map (the entire LR-NES was omitted) is drawn with a 1σ cutoff. www.nature.com/nature 8
Figure S8. Leptomycin B inhibits CRM1-SNUPN in the presence and absence of RanGTP. In vitro pull down assays of immobilized GST-SNUPN and CRM1 or CRM1 that was preincubated with leptomycin B, in the presence and absence of RanGTP. Bound proteins were visualized by SDS-PAGE and Coomassie staining. www.nature.com/nature 9
Figure S9. Mutations at CRM1-NES epitope II interface decrease CRM1-SNUPN interactions. a, In vitro pull down assays of immobilized GST-SNUPN, GST-SNUPN proteins mutated at NES epitope II and CRM1 in the presence and absence of RanGTP. b, In vitro pull down assays of immobilized GST-CRM1, GST-CRM1 mutated at its interface with NES epitope II of SNUPN and CRM1 in the presence and absence of RanGTP. Bound proteins were separated by SDS-PAGE and Coomassie stained. www.nature.com/nature 10
Figure S10. The convex surface of the C-terminal half of CRM1, from H13-H20, is acidic. The CRM1 interface for NES epitope II of SNUPN is outlined in black. Electrostatic surface potential of CRM1 is shown using a scale of -12 kt/e to +12 kt/e. www.nature.com/nature 11