1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Structural diversity of oligomeric β-propellers with different numbers of identical blades

  1. Evgenia Afanasieva
  2. Indronil Chaudhuri
  3. Jörg Martin
  4. Eva Hertle
  5. Astrid Ursinus
  6. Vikram Alva
  7. Marcus D Hartmann
  8. Andrei N Lupas  Is a corresponding author
  1. Max Planck Institute for Developmental Biology, Germany
Research Article
Cite this article as: eLife 2019;8:e49853 doi: 10.7554/eLife.49853
3 figures, 3 tables and 4 data sets

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.

https://doi.org/10.7554/eLife.49853.002
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.

https://doi.org/10.7554/eLife.49853.003
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.

https://doi.org/10.7554/eLife.49853.005
Figure 2—figure supplement 1
Sequences of WRAP constructs.

All constructs start with an additional GAMG after TEV-cleavage.

https://doi.org/10.7554/eLife.49853.006
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.

https://doi.org/10.7554/eLife.49853.007
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.

https://doi.org/10.7554/eLife.49853.008
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.

https://doi.org/10.7554/eLife.49853.009

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
28.8 kDa33.9 kDaTetramer52°C331 nm
313.6 kDa27.8 kDaDimer67°C335 nm
417.5 kDa32.7 kDaDimer63°C332 nm
521.8 kDa42.6 kDaDimer65°C333 nm
28.9 kDa28.4 kDaTetramer43°C345 nm
313.2 kDa39.5 kDaTrimer54°C341 nm
417.6 kDa26.7 kDaDimer65°C341 nm
522 kDa46.9 kDaDimer62°C341 nm
626.3 kDa107 kDaTetramer63°C340 nm
Table 2
Crystallization conditions and cryo protection
https://doi.org/10.7554/eLife.49853.010
ConstructProtein solutionReservoir solution (RS)Cryo solution
2-blades8 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-blades23 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-blades23 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-blades4 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 groupC2221P21P21212C2
Cell dimensions
a, b, c (Å)55.24, 119.7, 84.1553.55, 92.35, 61.4397.72, 127.2, 72.9639.75, 107.5, 179.2
α, β, γ (°)90.00, 90.00, 90.0090.00, 94.95, 90.0090.00, 90.00, 90.0090.00, 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) *
Rmerge4.8 (56.7)6.3 (89.4)11.0 (76.4)8.8 (45.4)
I / σI17.8 (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)
Redundancy4.31 (4.38)4.67 (4.44)3.70 (3.51)3.31 (3.40)
Refinement
Resolution (Å)32.3–1.7038.3–1.7538.7–2.1539.8–1.75
No. reflections29426569744740070952
Rwork/Rfree0.20/0.240.19/0.210.22/0.250.20/0.24
No. atoms
Protein2364535773505639
Ligands (Zn2+)0060
Water314302330691
B-factors
Protein24.3032.3036.5026.70
Ligands (Zn2+)--50.60-
Water35.3036.7033.7035.50
R.m.s. deviations
Bond lengths (Å)0.0120.0170.0110.013
Bond angles (°)1.551.721.511.53
  1. *Values in parentheses are for highest-resolution shell.

Data availability

Diffraction data have been deposited in PDB under the accession codes 6R5X, 6R5Z, 6R5Y, and 6R60.

The following data sets were generated
  1. 1
    Protein Data Bank
    1. E Afanasieva
    2. I Chaudhuri
    3. J Martin
    4. E Hertle
    5. A Ursinus
    6. V Alva
    7. MD Hartmann
    8. AN Lupas
    (2019)
    ID 6R5X. Structural diversity of oligomeric β-propellers with different numbers of identical blades.
  2. 2
    Protein Data Bank
    1. E Afanasieva
    2. I Chaudhuri
    3. J Martin
    4. E Hertle
    5. A Ursinus
    6. V Alva
    7. MD Hartmann
    8. AN Lupas
    (2019)
    ID 6R5Z. Structural diversity of oligomeric β-propellers with different numbers of identical blades.
  3. 3
    Protein Data Bank
    1. E Afanasieva
    2. I Chaudhuri
    3. J Martin
    4. E Hertle
    5. A Ursinus
    6. V Alva
    7. MD Hartmann
    8. AN Lupas
    (2019)
    ID 6R5Y. Structural diversity of oligomeric β-propellers with different numbers of identical blades.
  4. 4
    Protein Data Bank
    1. E Afanasieva
    2. I Chaudhuri
    3. J Martin
    4. E Hertle
    5. A Ursinus
    6. V Alva
    7. MD Hartmann
    8. AN Lupas
    (2019)
    ID 6R60. Structural diversity of oligomeric β-propellers with different numbers of identical blades.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)