(A) Top, domain organization of Alp14-monomer. Bottom, SEC-MALS for wt-Alp14-monomer at 100 mM KCl with and without DRP, revealing two αβ-tubulins bound in a non-polymerized state. (B) Top, domain …
We determined the molar ratio for four Alp14 constructs (described in Figure 1) in binding to soluble αβ-tubulins at 100 and 200 mM KCl conditions using quantitative-size exclusion chromatography …
For each condition, 1 μmol of each Alp14 construct (described in Figure 1) was mixed with defined molar amount of soluble αβ-tubulin (1–2 μmol moles per Alp14 subunit) and Darpin-D1 (DRP). (A, D) …
(A) Top and bottom panels: scheme for recombinant TOG1 and TOG2 domains studied and SDS-PAGE of purified 10 and 1 μM TOG1 and TOG2 domains, respectively. (B) Summary of measured αβ-tubulin binding …
(A–B) 3.6 Å X-ray crystal structure of the S. kluyveri 1:2:2 sk-Alp14:αβ-tubulin:DRP reveals pseudo-dimeric head-to-tail subunits (red and orange) in a TOG square assembly consisting of four TOG …
(A) Images of square crystals of 1:2:2 sk-Alp14-monomer:αβ-tubulin:DRP assemblies. (B) Molecular replacement search results for αβ-tubulin placement in the asymmetric unit. Note the distinct …
(A) Top scheme for Alp14, TOG1, TOG2, and linker sequence. Bottom, structure of TOG square with two TOG1 (blue) and two TOG2 (cyan) domains forming interfaces via inter-HEAT repeat elements with the …
(A) TOG square structure showing two cysteine pairs (green space fill) mutated in interfaces 1 (black-dashed lines) and 2 (red-dashed lines). (B) Close-up views of interfaces 1 (left) and 2 (right) …
(A) Mass-spectrometry-based strategy for disulfide peptide mapping for the sk-Alp14 S180C L304C mutant reveals that sequences of peptides in interface 1 form crosslinked disulfides, confirming that …
(A–D) Generation of structure-based TOG square assembly-defective mutants using wt-Alp14-dimer (A) through inactivation of interface 1 in the INT1 mutant (B: INT1, pink; eight mutant residues), …
(A) Purification of INT1 mutant. Top, SEC-elution profile and trace for INT1 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel show SDS-PAGE for SEC purified fractions. …
(A), (D), (G), and (J) Raw negative stain EM micrographs of WT-Alp14-dimer:αβ-tubulin, INT1, INT2, and INT1 +2, respectively. Note that the highlighted rectangle regions are shown in Figure 1. Boxes …
(A, B) Top, schemes of DRP and DRPΔN binding to αβ-tubulin. DRP shifts the equilibrium toward dissociation from αβ-tubulin. Bottom, isothermal titration calorimetery studies reveal a three-fold …
(A) SEC trace for the purified DRPΔN mutant. (B) SEC trace of 1:2:2 TOG1-TOG2:αβ-tubulin:DRPΔN complex (blue) in comparison to DRPΔN (red) and αβ-tubulin (black). (C) and (D) SDS-PAGE fractions …
(A) View of rectangular 1:2:1 TOG1-TOG2:αβ-tubulin:DRPΔN crystals formed in the same conditions as crystals in Figure Figure 2—figure supplement 1. (B) Top, molecular replacement solution to …
(A and B) Conformational change of TOG2 (blue) around TOG1 (red) while each is bound to αβ-tubulin (green and cyan) from a corner subunit in the wheel assembly (left) and in the extended …
(A) Right, atomic model for four αβ-tubulin-bound TOG square X-ray structures (Figure 2) docked using αβ-tubulin bound to TOG1 at the terminal αβ-tubulin in a curved protofilament (PDB ID: 3RYH). …
(A) An all-atom model for a TOG square bound to four αβ-tubulins docked onto the protofilament plus-end by overlaying the terminal αβ-tubulin with the αβ-tubulin bound onto TOG1. Note some minor …
An animation for this model is shown in Video 1. (A) Assembly of yeast MT polymerase dimeric TOG1-TOG2 subunits with four αβ-tubulins into an αβ-tubulin:TOG square. TOG squares diffuse along MT …
This animation describes the ‘polarized unfurling’ mechanism for multiple TOG domains in promoting MT polymerization. Briefly, Yeast MT polymerases are dimers with each subunit including TOG1 and …
Protein complex | Expected Mass (kDa) | SEC-MALS Measured Mass (kDa) | SEC elution volume (mL) | Apparent Mass (kDa) |
---|---|---|---|---|
αβ-Tubulin | 100 kDa | 98 ± 0.323* | 12.9 | 104 |
Alp14-dimer | 150 kDa | 143 ± 1.70 | 10.0 | 675 |
1 Alp14-dimer: 1 Tubulin 80 mM KCl | 350 kDa | 387 ± 2.74 | 9.27 | 463 |
1 Alp14-dimer: 2 Tubulin 80 mM KCl | 550 kDa | 578 ± 1.41 | 8.98 | 784 |
1 Alp14-dimer: 1Tubulin 200 mM KCl | 350 kDa | 304 ± 11.8 | 9.22 | 693 |
1 Alp14-dimer: 2 Tubulin 200 mM KCl | 350 kDa | 392 ± 9.66 | 9.67 | 549 |
1 Alp14-dimer-TOG1M: 1 Tubulin 80 mM KCl | 350 kDa | 400 ± 7.3 | 10.13 | 433 |
1 Alp14-dimer-TOG2M: 2 Tubulin 80 mM KCl | 350 kDa | 382 ± 14 | 9.76 | 524 |
Alp14-monomer | 62 kDa | 77.8 ± 1.21 | 12.98 | 99 |
1 Alp14-monomer: 2 Tubulin 80 mM KCl | 262 kDa | 264 ± 1.31 | 11.17 | 253 |
1 Alp14-monomer: 2 Tubulin: 2 DRP 80mM KCl | 298 kDa | 312 ± 2.32 | 10.94 | 285 |
1 Alp14-dimer-INT1: 2 Tubulin 80mM KCl | 550 kDa | 533 ± 3.11 | 8.95 | 775 |
1 Alp14-dimer-INT2: 2 Tubulin 80mM KCl | 550 kDa | 580 ± 3.11 | 8.90 | 770 |
1 Alp14-dimer-INT1+2: 2 Tubulin 80mM KCl | 550 kDa | 540 ± 3.22 | 8.80 | 790 |
*Standard error is defined based on fitting data across peaks using Astra-software.
Protein Complex (Alp14-monomer concentration 4.43 uM) | µM tubulin* bound | µM tubulin free | TOG1-TOG2 :αβ-tubulin |
---|---|---|---|
wt-Alp14-monomer (2 µM) | |||
1 Alp14-monomer: 1 Tub 100 mM KCl | 4.57 ± 0.08 | 0.43 ± 0.08 | 1.04 |
1 Alp14-monomer: 2 Tub 100 mM KCl | 8.82 ± 0.16 | 1.18 ± 0.16 | 2 |
1 Alp14-monomer: 1 Tub 200 mM KCl | 3.37 ± 0.27 | 1.63 ± 0.27 | 0.76 |
1 Alp14-monomer: 2 Tub 200 mM KCl | 5.97 ± 0.12 | 4.03 ± 0.12 | 1.35 |
1 Alp14-monomer:1 Tub:1 DRP 100 mM KCl | 4.33 ± 0.13 | 0.67 ± 0.13 | 0.98 |
1 Alp14-monomer:2 Tub:2 DRP 100 mM KCl | 7.23 ± 0.01 | 2.77 ± 0.01 | 1.64 |
1 Alp14-monomer:1 Tub:1 DRP 200 mM KCl | 4.13 ± 0.09 | 0.87 ± 0.09 | 0.94 |
1 Alp14-monomer: 2 Tub 2 DRP 200 mM KCl | 5.67 ± 0.03 | 4.34 ± 0.03 | 1.28 |
wt-Alp14-dimer (1 µM) | |||
1 Alp14-dimer: 1 Tub 100 mM KCl | 4.31 ± 0.07 | 0.69 ± 0.07 | 0.98 |
1 Alp14-dimer: 2 Tub 100 mM KCl | 7.11 ± 0.14 | 2.89 ± 0.14 | 1.61 |
1 Alp14-dimer: 1 Tub 200 mM KCl | 3.44 ± 0.37 | 1.56 ± 0.37 | 0.78 |
1 Alp14-dimer: 2 Tub 200 mM KCl | 5.46 ± 0.34 | 4.55 ± 0.34 | 1.24 |
1 Alp14-dimer: 1 Tub: 2 DRP 100 mM KCl | 4.23 ± 0.02 | 0.78 ± 0.02 | 0.96 |
1 Alp14-dimer: 1 Tub: 2 DRP 100 mM KCl | 8.33 ± 0.09 | 1.67 ± 0.09 | 1.89 |
1 Alp14-dimer: 1 Tub: 1 DRP 200 mM KCl | 4.17 ± 0.29 | 0.83 ± 0.29 | 0.95 |
1 Alp14-dimer: 2 Tub: 2 DRP 200 mM KCl | 6.02 ± 0.17 | 3.99 ± 0.17 | 1.36 |
TOG square Interface mutants (1 µM) | |||
1 INT1: 2 Tub 100mM KCl | 8.34 ± 0.07 | 1.66 ± 0.07 | 1.74 |
1 INT1: 2 Tub 200mM KCl | 5.67 ± 0.04 | 4.33 ± 0.04 | 1.18 |
1 INT2: 2 Tub 100mM KCl | 9.52 ± 0.3 | 0.48 ± 0.2 | 2.02 |
1 INT2: 2 Tub 200mM KCl | 6.82 ± 0.2 | 3.1 ±0.1 | 1.45 |
1 INT1+2: 2 Tub 100mM KCl | 8.44 ± 0.05 | 1.56 ± 0.05 | 1.86 |
1 INT1+2: 2 Tub 200mM KCl | 6.85 ± 0.04 | 3.15 ± 0.04 | 1.46 |
Inactivated TOG mutants (1 µM) | |||
1 TOG1M: 1 Tub 100 mM KCl | 4.04 ± 0.07 | 0.97 ± 0.07 | 0.92 |
1 TOG1M: 2 Tub 100 mM KCl | 4.94 ± 0.06 | 5.06 ± 0.06 | 1.12 |
1 TOG1M: 1 Tub 200 mM KCl | 1.66 ± 0.25 | 3.34 ± 0.25 | 0.38 |
1 TOG1M: 2 Tub 200 mM KCl | 2.23 ± 0.1 | 7.77 ± 0.1 | 0.51 |
1 TOG2M: 1 Tub 100 mM KCl | 3.53 ± 0.04 | 1.48 ± 0.04 | 0.8 |
1 TOG2M: 2 Tub 100 mM KCl | 6.24 ± 0.10 | 3.76 ± 0.1 | 1.41 |
1 TOG2M: 1 Tub 200 mM KCl | 3.15 ± 0.24 | 1.85 ± 0.24 | 0.72 |
1 TOG2M: 2 Tub 200 mM KCl | 4.28 ± 0.18 | 5.72 ± 0.18 | 0.97 |
*Standard error is defined based on combined data from duplicated SEC runs.
Data collection | 1:2:2 sk-Alp14-monomer: αβ-Tubulin:DRP | 1:2:2 sk-Alp14-monomer-SL: αβ-Tubulin:DRP | 1:2:2 sk-Alp14-monomer: αβ-Tubulin:DRPΔN | 1:2:2 sk-Alp14-dimer: αβ-Tubulin:DRPΔN |
---|---|---|---|---|
Resolution range (Å) | 96.59 - 4.40 (4.64- 4.40)* | 59.45 – 3.60 (3.79 – 3.60)* | 57.56 – 3.20 (3.37 – 3.20)* | 99.83 – 3.5 (3.69 – 3.50)* |
Space group | P 21 | P 21 | P 21 | P 21 |
Wavelength (Å) | 0.9792 | 0.9792 | 0.9792 | 0.9792 |
Unit cell (Å): a, b, c (°): β | 218.80, 107.65, 282.74 90.38 | 218.48, 106.15, 282.23 90.39 | 115.13, 194.99, 149.57 90.19 | 122.73 199.67, 162.70 90.09 |
Total number of observed reflections | 229567 | 380856 | 298551 | 235576 |
Unique reflections | 80099 {68039}† | 142673 {121943}† | 104265 {88337}† | 91368 |
Average mosaicity | 0.57 | 0.38 | 0.64 | 0.50 |
Multiplicity | 2.9 (2.9)* | 2.7 (2.7)* | 2.9 (2.9)* | 2.6 (2.4)* |
Completeness (%) | 95.4 (94.8) {80.6}† | 95.0 (96.7) {79.0}† | 96.2 (97.9) {82.0}† | 92.9 (90.2)* |
Wilson B-factor (Å2) | 82.7 | 81.4 | 46.6 | - |
<I/σ (I)> | 4.9 (1.9)* | 4.8 (1.2)* | 5.8 (1.5)* | 4.8 (1.1)* |
Rmergec | 0.14 (0.48)* | 0.13(0.65)* | 0.13(0.65)* | 0.14 (0.63)* |
Structure refinement | ||||
Rwork | 0.23 (0.26)* | 0.20 (0.24)* | 0.18 (0.23)* | - |
Rfree | 0.26 (0.33)* | 0.24 (0.26)* | 0.24 (0.26)* | - |
Complexes per asymmetric unit | 2 | 2 | 2 | - |
Number of atoms | 78030 | 77878 | 36865 | - |
Protein residues | 9989 | 9981 | 4661 | - |
Ligand atoms | 496 | 496 | 248 | - |
RMS bond lengths (Å) | 0.004 | 0.004 | 0.004 | - |
RMS bond angles (°) | 0.94 | 0.98 | 0.93 | - |
Ramachandran favored (%) | 94.0 | 94.0 | 95.0 | - |
Ramachandran allowed (%) | 5.4 | 5.5 | 4.5 | |
Ramachandran outliers (%) | 0.3 | 0.3 | 0.2 | - |
Clashscore | 4.5 | 4.8 | 5.6 | - |
Mean B values (Å2) | ||||
Overall | 108.4 | 98.3 | 48.6 | - |
Macromolecules | 108.6 | 98.4 | 48.6 | - |
Ligands | 78.5 | 91.5 | 36.4 | - |
*Numbers represent the highest-resolution shell.
†Numbers represent the truncated data after treated with ellipsoidal truncation and anisotropic scaling.
‡Rmerge = ΣhklΣi|Ii(hkl)-Iav(hkl)|/ΣhklΣiIi(hkl).
Interface | 1:2:2: sk-Alp14-monomer: αβ-Tubulin: DRP(Å2)* | 1:2:2: sk-Alp14-monomer-SL: αβ-Tubulin: DRP (Å2) | 1:2:1: sk-Alp14-monomer: αβ-Tubulin: DRP-ΔN (Å2) |
---|---|---|---|
TOG1 and TOG2-interface 2 | 273 ± 35 | 257 ± 42 | - |
TOG1-TOG2 dimer-interface 1 | 661 ± 27 | 702 ± 18 | - |
αβ-tubulin and TOG1 | 752 ± 12 | 786 ± 42 | 804 ± 24 |
αβ-tubulin and TOG2 | 916 ± 23 | 890 ± 22 | 863 ± 12 |
β-tubulin and DRP or DRP-ΔN | 842 ± 68 | 873 ± 21 | 846 ± 20 |
α-tubulin and DRP (inter-αβ-tubulin subunit) | 108 ± 47 | 81 ± 31 | - |
*Interface areas were determined by a single buried surface, and averaged among each non-crystallographic unit in the structure.
PDB ID | Intra dimer (α1β1) angle (°) | Inter dimer (β1α2) angle (°) | |
---|---|---|---|
Stathmin:RB3 complex with GTP | 3RYH | 9.2 | 10.3 |
Stathmin:RB3 complex with GDP | 1SA0 | 13.0 | 13.5 |
αβ-tubulin:TOG1 complex with GTP | 4FFB | 13 | - |
αβ-Tubulin:TOG2 complex with GTP | 4U3J | 13 | - |
αβ-Tubulin:DRP complex with GDP | 4DRX | 11.0 | - |
Sk-Alp14-monomer:αβ-Tubulin:DRP wheel with GDP | Current | 11.3 | - |
Sk-Alp14-monomer:αβ-Tubulin:DRP-ΔN with GDP | Current | 11.2 | 16.4 |
*Curvature of αβ-tubulin interface were determined as previously described by Rice and Brouhard 2015.
Reagent type (species) or resource | Designation | Source | Identifier | Additional information |
---|---|---|---|---|
Chemical compound, drug | Darpin D1 (Synthetic DNA) | Invitrogen | N/A | |
Chemical compound, drug | GTP | Sigma | G-8877 | |
Chemical compound, drug | GDP | Sigma | G7127 | |
Chemical compound, drug | Crystallization plates | TTP Labtech | 4150–05600 | |
Chemical compound, drug | Crystallization sparse matrix screens | Qiagen | N/A | |
Chemical compound, drug | PEG-8000 | Sigma | 1546605 | |
Chemical compound, drug | PEG-2000 | Sigma | 8.21037 | |
Chemical compound, drug | Copper(II) sulfate | Sigma | C1297 | |
Chemical compound, drug | 1, 10- phenanthroline | Sigma | 131377 | |
Chemical compound, drug | Trypsin | Sigma | T6567 | |
Chemical compound, drug | Chymotrypsin | Sigma | C6423 | |
Chemical compound, drug | Iodoacetamide | Sigma | I6125 | |
Chemical compound, drug | 4-Vinylpyridine | Sigma | V3204 | |
Other | 2:4:4 sk- Alp14-550:αβ- Tubulin:DRP | Protein Data Bank | PDB: #6MZF | Deposited Data (Atomic coordinates) |
Other | 2:4:4 sk-Alp14-550- SL:αβ-Tubulin: DRP | Protein Data Bank | PDB: #6MZE | Deposited Data (Atomic coordinates) |
Other | 1:2:1 sk-Alp14-550: αβ-Tubulin:DRPΔN | Protein Data Bank | PDB: #6MZG | Deposited Data (Atomic coordinates) |
Other | Saccharomyces cerevisiae Stu2p | UniprotKB/ Swiss-Prot | P46675 | Protein sequence |
Other | Saccharomyces kluyveri Stu2p or Alp14p | Lachancea kluyveri NRRL Y-12651 chromosome | SKLU-Cont10078 | Protein sequence |
Other | Schizosaccharomyces pombe Alp14p | UniprotKB/ Swiss-Prot | O94534 | Protein sequence |
Other | Chaetomium thermophilum Stu2 | UniprotKB/ Swiss-Prot | G0S3A7 | Protein sequence |
Other | SoluBL21 bacterial expression system | AmsBio | C700200 | Model system (expression system) |
Recombinant DNA reagent | pLIC_V2-Sc Stu2p-H6 | Current study | N/A | Recombinant DNA constructs Expressed in bacterial strains |
Recombinant DNA reagent | pLIC_V2-Sk Stu2p-H6 | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-Sc Stu2-550-H6 (TOG1-TOG2 monomer) | Al-Bassam et al., 2006 | ||
Recombinant DNA reagent | pLIC_V2-KL-Stu2 -monomer-H6 (residues 1–560) | Current study | NA | |
Recombinant DNA reagent | pLIC_V2-CT Stu2-mon omer-H6 (residues 1–550) | Current study | NA | |
Recombinant DNA reagent | pLIC_V2-SK Alp14-monomer- H6 (residues 1-550) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-Sk Alp14-monomer -SL-H6 (residues 1–550; linker residues replaced KL sequence; see Materials and methods) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2- sk-wt-Alp14- dimer-H6 (residues 1–724) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2 -sk-Alp14- dimer- H6 | Current study | N/A | |
Recombinant DNA reagent | S180C and L304C (residues 1–724) | |||
Recombinant DNA reagent | pLIC_V2- sk-Alp14- dimer- H6 | Current study | N/A | |
Recombinant DNA reagent | S41C and E518C (residues 1–724) | |||
Recombinant DNA reagent | pLIC_V2-wt -Alp14-dimer- H6 (residues 1–690) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-TOG1M - H6 (residues 1–690: Y23A and R23A) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-TOG2M - H6 (residues 1–690: Y300A and K381A) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-INT1-H6 (residues 1–690: L206A, L208A, F275R D276A, L277A, V278A, K320L, R359A) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-INT2-H6 (residues 1–690: L39D, S40A, D42A, L437D, S440A, E478A and R479A) | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2 -INT1 + 2 H6 (L206A, L208A, F275R D276A, L277A, V278A, K320L, R359A L39D, S40A, D42A, L437D, S440A, E478A and R479A) | Current study | N/A | |
Recombinant DNA reagent | pET303-H6-DRP | Current study | N/A | |
Recombinant DNA reagent | pLIC_V2-H6-DRPΔN | Current study | N/A | |
Other | αβ-tubulin purified from porcine brains | Current study | N/A | Native protein purification |
Other | αβ-tubulin purified from porcine brains | Castoldi and Popov, 2003 | ||
Software, algorithm | ASTRA V6.0 | Wyatt Technology | http://www.wyatt. com/products/ software/astra.html | |
Software, algorithm | NanoAnalyze | TA Instruments | http://www.tainstruments.com/ | |
Software, algorithm | EMAN2 | http://blake.bcm.edu/emanwiki/EMAN2 | ||
Software, algorithm | iMOSFLM | Battye et al., 2011 | http://www.mrc-lmb.cam.ac.uk/harry/imosflm/ver721/quickguide.html | |
Software, algorithm | PHASER | Terwilliger, 2000 | http://www.phaser.cimr.cam.ac.uk/index.php/ | |
Software, algorithm | PHASER | McCoy, 2007 | Phaser_Crystallographic_ Software | |
Software, algorithm | PyMol | Schrodinger, LLC | http://www.pymol.org/ | |
Software, algorithm | UCSF-Chimera | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ | |
Software, algorithm | DM from CCP4 suite | Cowtan and Main, 1996 | http://www.ccp4.ac.uk/html/dmmulti.html | |
Software, algorithm | PHENIX | Adams et al., 2010 | https://www.phenix-online.org | |
Software, algorithm | anisotropy server | Strong et al., 2006 | https://services.mbi.ucla.edu/anisoscale/ | |
Software, algorithm | Phyre protein homology model | Kelley et al., 2015 | www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index | |
Software, algorithm | Cr-yolo | Wagner et al., 2018 | http://sphire. mpg.de/wiki/ | |
Software, algorithm | Relion 2.2 | Kimanius et al., 2016 | https://www2.mrc-lmb.cam.ac.uk/relion/index.php | |
Software, algorithm | Cryosparc | Punjani et al., 2017 | https://cryosparc.com/ | |
Software, algorithm | MolProbity | Chen et al., 2012 | http://molprobity.biochem.duke.edu | |
Software, algorithm | Coot | Emsley et al., 2010 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
Software, algorithm | BLENDER 3D-animation | Blender foundation | https://www.blender.org/ |