1. Structural Biology and Molecular Biophysics

Structural model for differential cap maturation at growing microtubule ends

  1. Juan Estévez-Gallego
  2. Fernando Josa-Prado
  3. Siou Ku
  4. Ruben M Buey
  5. Francisco A Balaguer
  6. Andrea E Prota
  7. Daniel Lucena-Agell
  8. Christina Kamma-Lorger
  9. Toshiki Yagi
  10. Hiroyuki Iwamoto
  11. Laurence Duchesne
  12. Isabel Barasoain
  13. Michel O Steinmetz
  14. Denis Chrétien
  15. Shinji Kamimura
  16. J Fernando Díaz
  17. Maria A Oliva  Is a corresponding author
  1. Structural and Chemical Biology Department, Centro de Investigaciones Biológicas, CSIC, Spain
  2. Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de Rennes) – UMR 6290, France
  3. Departamento de Microbiología y Genética, Universidad de Salamanca-Campus Miguel de Unamuno, Spain
  4. Division of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institut, Switzerland
  5. ALBA synchrotron, CELLS, Spain
  6. Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Japan
  7. Diffraction and Scattering Division, Japan Synchrotron Radiation Research Institute, Japan
  8. University of Basel, Biozentrum, Switzerland
  9. Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Japan
https://doi.org/10.7554/eLife.50155
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Cite this article
  1. Juan Estévez-Gallego
  2. Fernando Josa-Prado
  3. Siou Ku
  4. Ruben M Buey
  5. Francisco A Balaguer
  6. Andrea E Prota
  7. Daniel Lucena-Agell
  8. Christina Kamma-Lorger
  9. Toshiki Yagi
  10. Hiroyuki Iwamoto
  11. Laurence Duchesne
  12. Isabel Barasoain
  13. Michel O Steinmetz
  14. Denis Chrétien
  15. Shinji Kamimura
  16. J Fernando Díaz
  17. Maria A Oliva
(2020)
Structural model for differential cap maturation at growing microtubule ends
eLife 9:e50155.
https://doi.org/10.7554/eLife.50155
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5 figures, 6 tables and 1 additional file

Figures

Figure 1
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Structure of tubulin bound to GDP-phosphate analogues.

(A) The T2R-TTL complex includes one RB3 molecule (orange), one TTL molecule (pink) and two tubulin heterodimers: α-tubulin (dark gray, GTP-bound, chains A and C), β-tubulin (light gray, GDP-bound, …

Figure 2 with 1 supplement
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Phosphate analogues sustain tubulin activation and MT stabilization.

(A) Time course assembly of 30 μM GDP-tubulin (gray line) with either 1 mM GTP (black line) or 1 mM GDP and increasing BeF3- concentrations (1 mM, 2 mM, 4 mM, 6 mM, 8 mM and 10 mM; from light to …

Figure 2—figure supplement 1
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Electron micrographs of MTs polymerized in the presence of BeF3- and AlFx.

(A) Electron micrograph of MTs grown in the presence of GDP-tubulin and 6 mM BeF3-. The inset shows length estimations of MTs (units are μm, n > 50) from samples containing 30 μM GDP-tubulin with 2 …

Figure 3 with 3 supplements
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Fiber diffraction of MT models systems.

GDP-BeF3--MT (blue), GDP-AlFx-MT (red), GMPPCP-MT (salmon), GMPCP-MT (yellow), GMPCPP-MT (orange), GDP-Tx-MT (brown) and GDP-MT (gray). (A) Top; representative image (GMPCPP-MTs) of meridional …

Figure 3—figure supplement 1
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Shear-flow aligned fiber diffraction experiments.

(A) 2X samples were incubated at 37°C and mixed (1:1) with pre-warmed 2% methylcellulose. The mixture was displayed onto a mica ring set on a ceramic holder and was inserted into the shear-flow …

Figure 3—figure supplement 2
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Shear-flow aligned fiber diffraction images.

Dataset images from MTs assembled under different conditions, showing the meridional diffraction. Insets centered on the equatorial diffraction highlight the presence (GDP-Tx-, GMPCPP- and …

Figure 3—figure supplement 3
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Shear-flow aligned fiber diffraction images of BeF3-- and AlFx-MTs in the presence of taxol.

All these dataset images show the presence of the 4 nm layer lines and the clear absence (inset) of the 8 nm layer line.

Figure 4 with 1 supplement
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Cryo-EM of GDP-, BeF3--and GMPCPP-MTs.

(A) Straightened images of microtubules with 13 PFs (N) and 3-start monomer helices (S), denoted 13_3 (N_S) MTs. For each condition: raw image (left) and filtered image using the J0 and JN layer …

Figure 4—figure supplement 1
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additional cryo-EM data.

(A) Turbidimetric analysis of MTs assembled in BRB80, 35°C, and in the presence of 1 mM GTP (tubulin concentration 40 μM, gray), 5 mM BeF3- (tubulin concentration 40 μM, dotted blue), 5 mM BeF3- in …

Figure 5
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Cap model derived from MT model systems.

GTP-bound state (BeF3-, blue), transition state (AlFx, red), expanded state (GMPCPP, GMPCP, GDP-Tx, orange), and GDP-bound state (gray). (A) Schematic GTPase related conformational changes within …

Tables

Table 1
Data collection and refinement statistics.
Native T2R-TTL-AlF3
(PDB 6s9e)		Native T2R-TTL-BeF3-
(PDB 6gze)
Data collection
Space groupP212121P212121
Cell dimensions
a, b, c (Å)104.999, 157.357, 180.261104.176, 156.744, 180.587
α, β, γ (°)90.00, 90.00, 90.0090.00, 90.00, 90.00
Resolution (Å)48.003–2.2549.458–2.49
Rmerge0.075 (1.222)0.071 (1.159)
Rpim0.025 (0.417)0.028 (0.473)
I/σI16.5 (1.8)7.1 (0.6)
Completeness (%)99.0 (99.0)100 (100)
Redundancy9.6 (9.2)7.1 (7.0)
CChalf0.979 (0.635)0.999 (0.993)
Refinement
Resolution (Å)48.003–2.2549.458–2.49
No. of reflections140102103915
Rwork/Rfree0.2029/0.22780.2121/0.2565
No. of atoms1770116799
Protein1727916572
Ligand223175
Water19952
B-factors
Protein64.080.4
Ligand59.573.0
Water45.767.5
Wilson B48.9064.70
r.m.s deviation
Bond lengths (Å)0.0020.003
Bond angles (°)0.5260.557
Ramachandran %
Favor/allow/out97.88/2.12/0.0097.52/2.48/0.00

  1. *Data were collected from a single crystal.

    **Values in parentheses are for the highest resolution shell.

Table 2
PDBePISA analysis of nucleotide-hydrogen bonding at the E-site.
Curved conformationStraight conformation
GTP
(5xp3)		GMPCPP
(3ryh)		GDP
(4i55)		BeF3-
(6gze)		AlF3
(6s9e)		GMPCPP
(3jat)		GMPCP
(3jal)		GDP
(3jar)		GTP-γ-S
(3jak)		GDP•Pi
(6evx)
Base and riboseQ11 S140 N206 N228S140 N228Q15 N206 N228N206 N228N206
N228		N206 Y224 N228S140 N206 Y224Q15 S140 N206 Y224Q15 S140 N206 Y224 N228S140, N206 N228
PαQ11 C12Q11 C12 S140C12C12C12Q11 C12Q11 C12 S140Q11 C12C12C12
PβQ11 G144 T145 G146Q11 T145 G146Q11 G144 T145 G146Q11 G144 T145 G146Q11
G144
T145
G146		Q11 G144 T145 G146Q11 G144 T145 G146Q11 G144 T145 G146Q11 G144 T145 G146Q11 G144 T145 G146
Pγ/
BeF3-/
AlF3/
Pi/		A99 G100 N101 G144 T145A99 G100 N101 G144 T145-A99 G100 N101 T145E71
N101
Pα		A99 G100 G144 T145--G144 T145T145
Mg2+yesyesyesyesyesyesnononono
Table 3
Fiber diffraction analysis of MTs in various nucleotide-bound states.
GDPGDP-TxGDP-BeF3-GTP-BeF3-GDP- AlFxGTP-AlFxGMPCPPGMPPCPGMPCP
radius (nm)11.42 ± 0.1010.87 ± 0.1011.21 ± 0.2511.16 ± 0.1011.25 ± 0.8411.18 ± 0.1211.63 ± 0.1011.62 ± 0.5911.75 ± 0.53
avg. PF number12.91 ± 0.1012.37 ± 0.1012.29 ± 0.2012.23 ± 0.1013.43 ± 1.1213.35 ± 0.1313.29 ± 0.0813.03 ± 0.9113.55 ± 0.45
inter-PF distances (nm)5.50 ± 0.035.45 ± 0.015.67 ± 0.095.67 ± 0.025.21 ± 0.465.22 ± 0.055.45 ± 0.035.55 ± 0.385.40 ± 0.02
avg. monomer length (nm)4.06 ± 0.014.18 ± 0.014.07 ± 0.014.07 ± 0.014.05 ± 0.054.05 ± 0.014.18 ± 0.014.06 ± 0.014.17 ± 0.01
1 nm band peak position (nm−1)6.19 ± 0.016.02 ± 0.016.17 ± 0.016.17 ± 0.016.20 ± 0.056.20 ± 0.016.02 ± 0.016.20 ± 0.016.03 ± 0.01

  1. *Values are Avg ± StdErr.

Table 4
Comparison between experimental and theoretical PF skew angles.
GDPBeF3-GMPCPP
MT type13_314_312_313_313_314_3
θexp+0.10 ± 0.21
(n = 15)		−0.62 ± 0.05
(n = 27)		+0.60 ± 0.05
(n = 52)		−0.27 ± 0.07
(n = 9)		+0.33 ± 0.11
(n = 12)		−0.51 ± 0.04
(n = 12)
θthe+0.05−0.74+0.61−0.31+0.37−0.44

  1. Theoretical PF skew angles (θthe) were calculated according to Equation 9, using a = 40.9 Å, r = 9.4 Å, and δx = 48.95 Å for GDP-MTs. For GMPCPP MTs, the monomer spacing a was increased to 42.1 Å, and for BeF3- the inter-PF subunit rise was increased to 9.7 Å.

Table 5
Taxol bound BeF3-- and AlFx-MTs.
GDP-BeF3-GTP-BeF3-GDP- AlFxGTP-AlFx
Average monomer length (nm)4.03 ± 0.014.03 ± 0.014.04 ± 0.054.04 ± 0.01
1 nm band peak position (nm−1)6.24 ± 0.016.24 ± 0.016.22 ± 0.056.22 ± 0.01
Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Biological SampleTubulin alphaUniprotP81947purified from calf-brain
Biological SampleTubulin betaUniprotQ6B856purified from calf-brain
Gene (Rattus norvergicus)Stathmin-4UniprotP63043Overexpression in E. coli
Gene (Gallus gallus)Tubulin-Tyrosine LigaseUniprotE1BQ43Overexpression in E. coli
Chemical compound, nucleotideGMPCPPJena BioscienceJena Bioscience:GpCpp- NU405
Chemical compound, nucleotideGMPPCPJena BioscienceJena Bioscience:GppCp
NU-402
Chemical compound, nucleotideGMPCPJena BioscienceJena Bioscience:GpCp
NU-414
Chemical compound, drugTaxolSigma AldrichSigma Aldrich:T7191
Software, algorithmXDShttp://xds.mpimf-heidelberg.mpg.de/RRID:SCR_015652
Software, algorithmAIMLESShttps://www.ccp4.ac.uk/RRID:SCR_015747
Software, algorithmPHASERhttps://www.phenix-online.org/documentation/reference/phaser.htmlRRID:SCR_014219
Software, algorithmPHENIXhttps://www.phenix-online.org/RRID:SCR_016736
Software, algorithmCOOThttps://www.ccp4.ac.uk/RRID:SCR_014222
Software, algorithmPDBePISAhttps://www.ebi.ac.uk/pdbe/pisa/RRID:SCR_015749
software, algorithmImageJhttps://imagej.nih.gov/ij/RRID:SCR_003070
Software, algorithmXRToolsBM26-DUBBLE, ESRF
Software, algorithmTubuleJhttps://team.
inria.fr/serpico/software/tubulej/

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