Structural determinants of nuclear export signal orientation in binding to exportin CRM1
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
hRio2NES and CPEB4NES bind CRM1 in orientation opposite to the PKINES.
(A) Six nuclear export signal (NES) consensus patterns (Φ is Leu, Val, Ile, Phe or Met; X is any amino acid). (B) Structure of PKINES (yellow cartoon) bound to Chromosome Region of Maintenance …
Figure 1—figure supplement 1
Electron densities of the wild-type NES peptides.
Stereo views of kicked OMIT map meshes contoured at the 3.0σ level, on the final, refined coordinates for (A) hRio2NES and (B) CPEB4NES as shown in sticks. Anomalous difference map for Se-met hRio2NE…
Figure 2
Comparison of plus and minus NESs.
Pairwise comparison of hRio2NES (blue) or CPEB4NES (purple) with PKINES (yellow; 3NBY) upon superposition of NES-bound CRM1 grooves. Hydrophobic NES residues (Φs) are shown as sticks and orientation …
Figure 3
Hydrophobic side chain preferences for hRio2NES binding to CRM1.
In vitro pull-down assay (Coomassie-stained SDS/PAGE) of purified human CRM1 binding to immobilized GST-hRio2NES mutants (A) Φ3, Φ4, or Φ5 or (B) Φ1 or Φ2 position mutated in the presence of excess S…
Figure 4 with 2 supplements
hRio2NES, CPEB4NES, and their reverse counterparts bind CRM1 with similar binding affinities.
(A) Sequences of NESs used. (B) Binding of FITC-PKINES and various MBP-NESs to CRM1 measured by differential bleaching, monitored by a microscale thermophoresis instrument. MBP-NESs compete with …
Figure 4—figure supplement 1
Binding affinities of shorter hRio2NES and CPEB4NES constructs.
(A) Sequences of shorter hRio2NES and CPEB4NES constructs. (B) Binding of these NESs to CRM1 measured by differential bleaching. Comparison of hRio2NES constructs in Figure 4 and in this supplement …
Figure 4—figure supplement 2
Differential bleaching of fluorescence probe in a sigmoidal and binding-dependent manner.
Fluorogram of normalized fluorescence signal collected from the direct titration of CRM1 into fluorescent-labeled PKINES peptide in presence of excess ScRanGTP is plotted on the top panel. Traces …
Figure 5 with 1 supplement
hRio2NES-R and CPEB4NES-R are plus NESs.
(A) Structures of hRio2NES-R (light blue) and CPEB4NES-R (light pink) bound to CRM1 (gray surfaces). (B) Pairwise comparisons of PKINES (yellow), hRio2NES-R (light blue), and hRio2NES (blue) when …
Figure 5—figure supplement 1
Electron densities of the reverse NES peptides.
Stereo views of kicked OMIT map meshes, on the final coordinates for (A) hRio2NES-R, (B) CPEB4NES-R and as shown in sticks. Anomalous difference map for Se-met hRio2NES-R is contoured at the 3.0σ …
Figure 6 with 1 supplement
N-terminal ΦXΦ motif generates a minus orientation PKINES-Flip mutant.
(A) Sequence alignment of PKINES and PKINES-Flip peptides with their hydrophobic residues of ΦXΦ motifs in red and the NES helix shown as a cylinder. (B) Pull-down assay of immobilized GST-PKINES …
Figure 6—figure supplement 1
Electron densities of the PKINES-Flip3 NES peptide.
Stereo views of kicked OMIT map meshes, on the final coordinates for PKINES-Flip3.
Figure 7
Prevalence of putative minus NESs in the Dbase data set.
(A) Consensus patterns for minus NESs in new NES classes 1a-R to 1d-R (reverse of class 1 patterns). (B) The number of sequences in the 246 proteins in Dbase that match the class 1 (+) and class 1-R …
Figure 8
Putative minus NESs in the Dbase data set.
(A) Summary of putative minus NESs (in the Dbase data set that match class 1-R patterns exclusively) tested for CRM1 binding. Nap1p (*) was previously shown to direct nuclear export in cells even …
Tables
Table 1
Data collection and refinement statistics
| ScXPO1-RanGppNHp-Yrb1p bound to NES of: | |||||
|---|---|---|---|---|---|
| Selenomethione-hRio2 | CPEB4 | Selenomethione-hRio2-R | CPEB4-R | PKI-Flip3 | |
| Data collection | |||||
| Space group | P43212 | ||||
| Cell dimensions | |||||
| a, b, c (Å) | 106.48, 106.48, 303.73 | 105.96, 105.96, 304.00 | 106.69, 106.69, 304.50 | 106.48, 106.48, 303.73 | 105.96, 105.96, 304.00 |
| a, b, g (°) | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 |
| Resolution (Å) | 50.00–2.28 (2.32–2.28)* | 50.00–2.10 (2.14–2.10) | 50.00–2.28 (2.32–2.28) | 50.00–2.94 (3.00–2.94) | 50.00–2.55 (2.59–2.55) |
| Rpim | 2.9 (37.7) | 3.5 (43.4) | 3.5 (38.6) | 4.9 (40.6) | 4.1 (46.5) |
| I/sI | 24.3 (2.17) | 19.5 (1.70) | 22.5 (2.72) | 13.3 (1.87) | 19.0 (1.92) |
| Completeness (%) | 98.6 (99.8) | 99.5(100) | 98.0 (99.2) | 94.6 (96.0) | 99.6 (100) |
| Redundancy | 7.0 (5.9) | 6.0 (6.1) | 7.0 (7.0) | 6.2 (5.7) | 5.5 (5.5) |
| Refinement | |||||
| Resolution (Å) | 45.7–2.28 (2.32–2.28) | 40.2–2.09 (2.12–2.09) | 37.7–2.28 (2.31–2.28) | 47.5–2.94 (3.02–2.94) | 47.5–2.54 (2.60–2.54) |
| No. reflections | 77,245 (2833) | 98,659 (1793) | 79,492 (3267) | 34,265 (2013) | 56862 (3361) |
| Rwork/Rfree | 17.8 (25.8)/21.9 (27.3) | 17.0 (23.8)/20.8 (27.0) | 16.8 (24.7)/21.2 (27.6) | 18.1 (25.2)/24.0 (31.3) | 18.6 (25.0)/22.6 (30.6) |
| No. atoms | |||||
| Protein | 10,859 | 11,114 | 10,823 | 10,708 | 10797 |
| Ligand/ion | 60 | 76 | 59 | 51 | 51 |
| Water | 271 | 660 | 358 | 8 | 253 |
| NES Peptide/Φ | 111/46 | 122/43 | 130/46 | 112/43 | 105/43 |
| B-factors | |||||
| Protein | 42.0 | 39.3 | 43.9 | 53.8 | 46.5 |
| Ligand/ion | 44.3 | 51.7 | 46.9 | 41.8 | 41.6 |
| Water | 33.4 | 34.8 | 35.4 | 23.3 | 35.3 |
| NES peptide/Φ | 80.5/77.3 | 77.6/70.4 | 67.5/61.7 | 81.2/80.5 | 98.6/96.0 |
| R.m.s deviations | |||||
| Bond lengths (Å) | 0.003 | 0.003 | 0.006 | 0.003 | 0.004 |
| Bond angles (°) | 0.617 | 0.689 | 0.835 | 0.578 | 0.673 |
| PDB code | 5DHF | 5DIF | 5DI9 | 5DHA | 5DH9 |
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*
Highest resolution shell is shown in parenthesis.
-
One crystal was used for each structure.
