Structural diversity of oligomeric β-propellers with different numbers of identical blades
- Max Planck Institute for Developmental Biology, Germany
Figures
Figure 1 with 1 supplement
The 7-bladed propeller in the protein PkwA of Thermomonospora curvata.
(A) Multiple sequence alignment of the seven blades and sequences of the constructs used to test the formation of higher-order oligomers in vitro. Non-identical residues in the repeats are colored red. The four β−strands of the propeller blades are indicated above the alignment. (B) Crystal structure of the PkwA propeller (PDB 5YZV). The structure is colored in rainbow colors from blue at the N-terminus to red at the C-terminus. The velcro closure resulting from the last strand of the last blade being permuted to the N-terminus is clearly visible. (C) Oligomerization of PkwA consensus repeats. Differentiation of propeller sizes was achieved by native polyacrylamide gel electrophoresis. Lanes 8–10 show migration of homo-oligomeric propeller complexes assembled from 3-, 4- and 5-bladed repeats. Lanes 1–7 show mixtures of different building blocks to probe for hetero-oligomeric assembly. Proteins were mixed in equimolar ratios (lanes 3–7), unfolded and refolded together. For mixtures of 2- and 3-bladed repeats (lanes 1 and 2) 2:1 molar ratios were used. In all cases, regardless of the mixture composition, PkwA repeats re-assembled only into homo-oligomers.
Figure 1—figure supplement 1
Sequences of PkwA constructs.
Due to the thrombin cleavage site motif, all PkwA proteins originally expressed as GST-fusions start with an additional GS and proteins expressed with a His6-tag start with GSHM.
Figure 2 with 2 supplements
The recently amplified WRAP propeller in Npun_R6612 of Nostoc punctiforme PCC73102.
(A) Multiple sequence alignment of the 14 blades of WRAP. Non-identical repeats are colored in red and the non-repeating β-strand 4 of the velcro blade is underlined. The four β-strands of the propeller blades are indicated above the alignment. The repeat unit chosen for in vitro studies is highlighted by a box. (B) Crystal structure of the WRAP propeller (PDB 2YMU). (C) Oligomerization of WRAP repeats. Assembly was probed by crosslinking proteins with 0.6% glutaraldehyde (GA) and subsequent analysis by SDS-PAGE and Coomassie Blue G250 staining. On the left side, non-crosslinked proteins are shown for comparison.
Figure 2—figure supplement 1
Sequences of WRAP constructs.
All constructs start with an additional GAMG after TEV-cleavage.
Figure 2—figure supplement 2
Purification of WRAP fragments.
Shown are the elution profiles of the Superdex 75 10/300 gel size-exclusion column runs.
Figure 3 with 1 supplement
Structures of homo-oligomeric WRAP propellers.
Subunits in each propeller are colored in rainbow colors with blue at the N-terminus and red at the C-terminus. (A) 8-bladed propeller formed of four 2-bladed fragments (PDB 6R5X). (B) 9-bladed propeller formed of three 3-bladed fragments (6R5Z). (C) 8-bladed propeller formed of two 4-bladed fragments (6R5Y). (D) 9-bladed asymmetric fold formed of two 5-bladed fragments (6R60). (E) Superimposition of all intra-subunit interfaces in 2-, 3-, 4-, and 5-bladed fragments. (F) Superimposition of all inter-subunit interfaces in the two 8-bladed propellers and the 9-bladed propeller.
Figure 3—figure supplement 1
Truncation of 5-bladed WRAP repeats correlates with an asymmetric, incomplete propeller structure.
Crystals of the dimerized 5-bladed His6-tagged WRAP repeat were dissolved and analyzed by SDS-PAGE and Coomassie Blue G250 staining (lane 3). For comparison, native (lane 1) and His6-tagged (lane 2) 5-bladed repeats are shown. In lane 4, protein from a clear crystallization solution that gave no crystals is loaded. The resulting four bands of lane 3 (boxed) were analyzed by mass spectrometry and shown to contain protein species with an intact N-terminal blade (upper two bands), and protein species were the first blade is largely missing (lower two bands; underlined part in the sequence of the 5-bladed fragment). The four β−strands in an intact WRAP blade are indicated above the alignment.
Tables
Table 1
Summary of biophysical data for the different propeller constructs of PkwA (upper panel) and WRAP (lower panel).
https://doi.org/10.7554/eLife.49853.004| Propeller Blades in protomer | Molecular mass protomer calculated | Molecular mass SLS measured | Assembly state based on SLS | CD melting temperature Tm | Tryptophan fluorescence λmax |
|---|---|---|---|---|---|
| 2 | 8.8 kDa | 33.9 kDa | Tetramer | 52°C | 331 nm |
| 3 | 13.6 kDa | 27.8 kDa | Dimer | 67°C | 335 nm |
| 4 | 17.5 kDa | 32.7 kDa | Dimer | 63°C | 332 nm |
| 5 | 21.8 kDa | 42.6 kDa | Dimer | 65°C | 333 nm |
| 2 | 8.9 kDa | 28.4 kDa | Tetramer | 43°C | 345 nm |
| 3 | 13.2 kDa | 39.5 kDa | Trimer | 54°C | 341 nm |
| 4 | 17.6 kDa | 26.7 kDa | Dimer | 65°C | 341 nm |
| 5 | 22 kDa | 46.9 kDa | Dimer | 62°C | 341 nm |
| 6 | 26.3 kDa | 107 kDa | Tetramer | 63°C | 340 nm |
Table 2
Crystallization conditions and cryo protection
https://doi.org/10.7554/eLife.49853.010| Construct | Protein solution | Reservoir solution (RS) | Cryo solution |
|---|---|---|---|
| 2-blades | 8 mg/ml protein 50 mM TRIS HCl pH 8.0 150 mM sodium chloride | 200 mM sodium acetate 100 mM TRIS HCl pH 8.5 30%(w/v) PEG 4000 | n/a |
| 3-blades | 23 mg/ml protein 50 mM TRIS HCl pH 7.5 150 mM sodium chloride | 200 mM ammonium fluoride 20%(w/v) PEG 3350 | RS + 10%(v/v) PEG 400 |
| 4-blades | 23 mg/ml protein 50 mM TRIS HCl pH 7.5 150 mM sodium chloride | 10 mM zinc chloride 100 mM Hepes pH 7.0 20%(w/v) PEG 6000 | RS + 10%(v/v) PEG 400 |
| 5-blades | 4 mg/ml protein 50 mM HEPES pH 7.5 100 mM sodium chloride | 100 mM Magnesium chloride 100 mM HEPES pH 7.0 15%(w/v) PEG 4000 | RS + 15%(v/v) PEG 400 |
Table 3
Crystallographic data collection and refinement statistics
https://doi.org/10.7554/eLife.49853.011| Construct (PDB ID) | 2-blades (6R5X) | 3-blades (6R5Z) | 4-blades (6R5Y) | 5-blades (6R60) |
|---|---|---|---|---|
| Data collection | ||||
| Space group | C2221 | P21 | P21212 | C2 |
| Cell dimensions | ||||
| a, b, c (Å) | 55.24, 119.7, 84.15 | 53.55, 92.35, 61.43 | 97.72, 127.2, 72.96 | 39.75, 107.5, 179.2 |
| α, β, γ (°) | 90.00, 90.00, 90.00 | 90.00, 94.95, 90.00 | 90.00, 90.00, 90.00 | 90.00, 94.40, 90.00 |
| Resolution (Å) | 32.3–1.70 (1.80–1.70) * | 38.3–1.75 (1.85–1.75) * | 38.7–2.15 (2.28–2.15) * | 39.8–1.75 (1.85–1.75) * |
| Rmerge | 4.8 (56.7) | 6.3 (89.4) | 11.0 (76.4) | 8.8 (45.4) |
| I / σI | 17.8 (2.32) | 13.5 (1.55) | 10.1 (1.94) | 9.17 (1.92) |
| Completeness (%) | 99.3 (97.4) | 99.2 (95.9) | 99.4 (99.0) | 98.1 (95.3) |
| Redundancy | 4.31 (4.38) | 4.67 (4.44) | 3.70 (3.51) | 3.31 (3.40) |
| Refinement | ||||
| Resolution (Å) | 32.3–1.70 | 38.3–1.75 | 38.7–2.15 | 39.8–1.75 |
| No. reflections | 29426 | 56974 | 47400 | 70952 |
| Rwork/Rfree | 0.20/0.24 | 0.19/0.21 | 0.22/0.25 | 0.20/0.24 |
| No. atoms | ||||
| Protein | 2364 | 5357 | 7350 | 5639 |
| Ligands (Zn2+) | 0 | 0 | 6 | 0 |
| Water | 314 | 302 | 330 | 691 |
| B-factors | ||||
| Protein | 24.30 | 32.30 | 36.50 | 26.70 |
| Ligands (Zn2+) | - | - | 50.60 | - |
| Water | 35.30 | 36.70 | 33.70 | 35.50 |
| R.m.s. deviations | ||||
| Bond lengths (Å) | 0.012 | 0.017 | 0.011 | 0.013 |
| Bond angles (°) | 1.55 | 1.72 | 1.51 | 1.53 |
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*Values in parentheses are for highest-resolution shell.
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Structural diversity of oligomeric β-propellers with different numbers of identical blades
eLife 8:e49853.
https://doi.org/10.7554/eLife.49853
