Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain
- Department of Chemistry, University of Calgary, Canada
- Department of Biology, McGill University, Canada
- Department of Biochemistry and Molecular Biology, University of Calgary, Canada
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, United States
- Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, United States
Figures
Figure 1 with 1 supplement
Doublecortin (DCX) structure and lattice interaction options.
(A) The disordered regions of DCX sequence predicted using PrDOS (Ishida and Kinoshita, 2007). (B) Schematic representations of the four orientational challenges to the elucidation of the …
Figure 1—figure supplement 1
The evaluation of purity and activity of in house purified Doublecortin.
(A) The purity of DCX-Light and DCX-Heavy isotopically labeled sample was evaluated on 8% SDS-PAGE gel. (B) Turbidity measurement at 340 nm versus time for Tubulin (10 μM) with different …
Figure 2 with 2 supplements
Crosslinking mass spectrometry analysis of Doublecortin (DCX).
(A) Two-dimensional crosslinking map linking α−β-tubulin and DCX at specific residues. NDC and C-terminal DC (CDC) domains of DCX sequence are shown with red and cyan, respectively. Intraprotein …
Figure 2—figure supplement 1
15N incorporation in purified heavy labeled DCX was assessed by LC-MS analysis.
The two MS1 scans for two candidate DCX peptides are shown as the examples. The highlighted mass to charge ratios (M/Zs) are corresponding to a range of species from fully 14N to fully 15N species. …
Figure 2—figure supplement 2
Monitoring the ratio of heterogenous species (Heavy-Light:HL and Light-Heavy:LH) to the homogenous species (Light-Light:LL and Heavy-Heavy:HH), for different crosslinked peptides.
The X-axis IDs are crosslinked residues. The experimental procedure for each sample is schematically represented in right. Only two peptides were shared among different samples (221-193 an 264-345). …
Figure 3
Integrative structure determination of microtubule (MT)–Doublecortin (DCX), using four stage workflow.
(1) Data gathering, including chemical crosslinking, atomic structures (MT structure, PDB 6EVZ; NDC structure, DCX component of PDB 4ATU; CDC structure, PDB 5IP4), and cryo-EM map EMD 2095. (2) …
Figure 4 with 4 supplements
Integrative structural modeling for NDC/CDC–microtubule (MT).
(A) The centroid structure for the main cluster of models produced by IMP for NDC–MT guided exclusively by crosslinking mass spectrometry (XL-MS) restraints. (B) The centroid structure for the main …
Figure 4—figure supplement 1
IMP analysis output for MT-NDC.
(a) The distribution of scores for the models computed for MT-NDC, using only crosslinking data (500,000 models). (b) The number of models randomly selected for model samples A (yellow) and sample B …
Figure 4—figure supplement 2
IMP analysis output for MT-CDC.
(a) The distribution of scores for the models computed for MT-CDC, using only crosslinking data (500,000 models). (b) The number of models randomly selected for model samples A (yellow) and B (red), …
Figure 4—figure supplement 3
Integrative structural modeling for NDC-MT and CDC-MT (2nd major clusters).
The centroid structure for the 2nd major cluster of models produced by IMP for NDC-MT (A) and CDC-MT (B) guided exclusively by XL-MS restrains; alpha-Tubulin and beta-Tubulin are shown as light and …
Figure 4—figure supplement 4
Crosslinking-mass spectrometry analysis of DCX-MT using conventional crosslinkers.
Two-dimensional crosslinking map linking alpha/beta-Tubulin and DCX at specific residues using (A) DSS and (B) EDC. NDC and CDC domains of DCX sequence are shown with red and cyan, respectively. A …
Figure 5 with 2 supplements
Integrative modeling of Doublecortin (DCX) self-assembly on microtubule (MT) lattice.
(A) The dimeric DCX–MT centroid model of the main clusters of models generated using only DCX–DCX crosslinking restraints. Four different relative positions of fixed NDC on the MT lattice were …
Figure 5—figure supplement 1
IMP analysis output for MT-dimeric DCX using DCX-DCX crosslinking data, where the NDCs were fixed at lateral (A), longitudinal (B), diagonal 1 (C) and diagonal 2 (D) relative positions on the MT lattice.
(a) The distribution of scores for the models computed for MT-dimeric DCX using the globular CDC structure (100,000 models). (b) The number of models randomly selected for model samples A (yellow) …
Figure 5—figure supplement 2
IMP analysis output for MT-dimeric DCX using MT-DCX and DCX-DCX crosslinking data, where the NDCs were fixed at lateral (A), longitudinal (B), diagonal 1 (C) and diagonal 2 (D) relative positions on MT lattice.
(a) The distribution of scores for the models computed for the MT-dimeric DCX using globular CDC structure (320,000 models). (b) The number of models randomly selected for model samples A (yellow) …
Figure 6
Positional evaluation of dimeric Doublecortin (DCX) on microtubule (MT) lattice.
(A) The fractional XL satisfaction for the main cluster of models generated for dimeric DCX–MT, employing all crosslinking restraints. The four different relative positions of fixed NDC on the MT …
Figure 7
Cooperative binding of C-terminal tail truncated Doublecortin (DCX) on microtubule (MT).
(A) Schematic of DCX-mCherry engaging in cooperative binding with DCX-GFP or DCX-R303X-GFP on the MT lattice. (B) Images of taxol MTs, 0.5 nM DCX-GFP, 2.5 nM DCX-mCherry, and the color-combined …
Figure 8
Mechanism of Doublecortin (DCX)-mediated microtubule (MT) nucleation and stabilization.
DCX stabilizes early GTP-tubulin oligomers through the CDC domain, which is then replaced with the NDC domain at the canonical binding site as during full MT assembly. NDC binding triggers a …
Author response image 1
(A) Crosslink sites on the MT lattice repeat unit highlighted in blue, showing that some are indeed buried within the interprotofilament groove.
(B) Alternative representation showing the buried nature of NDC on the lattice.
Author response image 2
Structural alignment of the new MT-CDC structure (6RF2) to the one used in our study (5IP4), placed at the NDC binding site for illustration.
CDC structures corresponding to 6RF2 and 5IP4 are shown with blue and cyan, respectively, α tubulins are shown in light grey and β tubulins are shown in dark grey, The RMSD calculated for residues …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | DCX | UniProtKB | O43602 | |
| Gene (Sus scrofa) | α-Tubulin | UniProtKB | P02550 | |
| Gene (Sus scrofa) | β-Tubulin | UniProtKB | P02554 | |
| Strain, strain background (Escherichia coli) | Arctic Express (DE3) | Agilent | 230,192 | Electrocompetent cells |
| Strain, strain background (Escherichia coli) | BL21(DE3) | New England BioLabs Inc. | C2527 | Mix and Go competent cells |
| Antibody | Anti-His (Mouse monoclonal) | Applied Biological Materials | G020 | WB (1:1000) |
| Antibody | Anti-β-tubulin (Mouse monoclonal) | Sigma-Aldrich | T4026 | (1:20 dilution in BRB80) |
| Recombinant DNA reagent | DCX-WT-pHAT-HUS | Gift of Dr. Susanne Bechstedt | Human doublecortin (1–365) plasmid | |
| Recombinant DNA reagent | DCX-WT-pHAT-HUGS | Gift of Dr. Susanne Bechstedt | GFP version of Human doublecortin (1–365) plasmid | |
| Recombinant DNA reagent | DCX-R303X-pHAT-HUGS | Gift of Dr. Susanne Bechstedt | GFP version of Human doublecortin (1–302) plasmid | |
| Recombinant DNA reagent | DCX-WT-pHAT-HUCS | Gift of Dr. Susanne Bechstedt | mCherry version of Human doublecortin (1–365) plasmid | |
| Peptide, recombinant protein | Human doublecortin (1–365) | This paper | Purified from E. coli Arctic Express cells | |
| Peptide, recombinant protein | GFP-doublecortin (1–365) | This paper | Purified from E. coli BL21 cells | |
| Peptide, recombinant protein | GFP-doublecortin (1-302) | This paper | Purified from E. coli BL21 cells | |
| Peptide, recombinant protein | mCherry-doublecortin (1-365) | This paper | Purified from E. coli BL21 cells | |
| Peptide, recombinant protein | α/β-Tubulin | Cytoskeleton | TL590M-A | |
| Peptide, recombinant protein | Rhodamine-labeled α/β-tubulin | Cytoskeleton | T240 | |
| Peptide, recombinant protein | Streptavidin–HRP | Thermo Fisher Scientific | N100 | |
| Peptide, recombinant protein | Glucose oxidase | Sigma-Aldrich | G2133-10KU | |
| Peptide, recombinant protein | Catalase | Sigma-Aldrich | E3289 | |
| Chemical compound, drug | Paclitaxel | European Pharmacopoeia Reference Standard | Y0000698 | |
| Chemical compound, drug | Docetaxel | Sigma-Aldrich | 01885 | |
| Chemical compound, drug | Atto 633 NHS-ester | ATTO-TEC GmbH | AD 633-35 | |
| Chemical compound, drug | LC-SDA | Thermo Fisher Scientific | 26,168 | |
| Chemical compound, drug | DSS | Thermo Fisher Scientific | 21,655 | |
| Chemical compound, drug | EDC | Thermo Fisher Scientific | 22,980 | |
| Software, algorithm | xVis | https://xvis.genzentrum.lmu.de/login.php | PMID:25956653 | |
| Software, algorithm | xiNET | http://crosslinkviewer.org/ | PMID:25648531 | |
| Software, algorithm | IMP | https://integrativemodeling.org/ | v.2.12 | PMID:22272186 |
| Software, algorithm | Mass Spec Studio | https://www.msstudio.ca | V2.0 | PMID:25242457 |
| Software, algorithm | TrackMate | https://imagej.net/plugins/trackmate/ | PMID:27713081 |
Additional files
-
Supplementary file 1
Complete list of crosslinking sites (and their associated crosslinked peptides) identified for MT-DCX construct using LC-SDA photo-chemical crosslinking.
- https://cdn.elifesciences.org/articles/66975/elife-66975-supp1-v2.xlsx
-
Supplementary file 2
The analysis output of all integrative structural modeling runs.
Column titles with star are defined at the bottom of the table.
- https://cdn.elifesciences.org/articles/66975/elife-66975-supp2-v2.xlsx
-
Supplementary file 3
Complete list of unique DCX-DCX crosslinking sites (and their associated crosslinked peptides) identified using isotope-assisted chemical crosslinking.
- https://cdn.elifesciences.org/articles/66975/elife-66975-supp3-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/66975/elife-66975-transrepform1-v2.pdf
