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A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

Nature Structural & Molecular Biology volume 24, pages 931–943 (2017)Cite this article

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An Author Correction to this article was published on 30 April 2020

Abstract

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

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Figure 1: The CKK is a highly conserved domain for MT minus-end tracking.
Figure 2: The unique MT-binding site of the CAMSAP CKK domain.
Figure 3: The interaction with four tubulin monomers is distributed across the CKK domain.
Figure 4: Validation of CKK–MT contact sites with in vitro assays and structure of a mutant CKK bound to MTs.
Figure 5: Examining CKK's preferred tubulin conformation.
Figure 6: CAMSAP CKKs protect MT minus ends from MCAK-induced depolymerization via steric inhibition.
Figure 7: Proposed mechanisms of MT minus-end binding and protection from MCAK-induced depolymerization by the CKK domain.

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Acknowledgements

We thank F. Govers (Wageningen University), V. Gelfand (Northwestern University) and M. Harterink (Utrecht University) for sharing reagents; E. Katrukha for advice on processing fluorescence microscopy data; R. Boelens for providing access to the solution-state NMR instrumentation; and S. Yokoyama and A. Nomura for sharing NMR resonance assignments of free CKK. A.A. was supported by ERC Synergy grant 609822. J.A., C.A.M. (MR/J000973/1), A.-P.J. and M.T. (MR/M019292/1) were supported by the Medical Research Council, UK. A.J.R. was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and Royal Society (104196/Z/14/Z). G.S. and B.J.G. were supported by the National Institutes of Health (R01GM070862). M.B., Y.L. (grant 718.015.001) and K.H. (grant 718.015.001 and 184.032.207) were supported by the Netherlands Organization for Scientific Research (NWO). ssNMR experiments were supported by uNMR-NL, an NWO-funded National Roadmap Large-scale Facility of the Netherlands (grant 184.032.207). M.O.S. was supported by grants from the Swiss National Science Foundation (31003A_166608) and from SystemsX.ch (RTD-TubeX).

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Author notes

  1. Joseph Atherton and Kai Jiang: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Structural and Molecular Biology, Birkbeck, University of London, London, UK

    Joseph Atherton, Agnel-Praveen Joseph, Anthony J Roberts, Maya Topf & Carolyn A Moores

  2. Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands

    Kai Jiang, Shasha Hua & Anna Akhmanova

  3. Division of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland

    Marcel M Stangier & Michel O Steinmetz

  4. NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands

    Yanzhang Luo, Klaartje Houben & Marc Baldus

  5. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands

    Jolien J E van Hooff

  6. Department of Biology, Theoretical Biology and Bioinformatics, Faculty of Science, Utrecht University, Utrecht, The Netherlands

    Jolien J E van Hooff

  7. Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands

    Jolien J E van Hooff

  8. Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Guido Scarabelli

  9. Division of Biological Sciences, University of California San Diego, La Jolla, California, USA

    Barry J Grant

  10. University of Basel, Biozentrum, Basel, Switzerland

    Michel O Steinmetz

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  1. Joseph Atherton
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Contributions

J.A., K.J., M.O.S., M.B., C.A.M. and A.A. designed experiments, analyzed data and wrote the paper. C.A.M. and A.A. coordinated the project. J.A. performed and analyzed cryo-EM and cryo-ET experiments; K.J. and S.H. performed in vitro reconstitution experiments; M.M.S. performed X-ray crystallography and biophysical experiments; Y.L. and K.H. performed and analyzed NMR experiments; J.J.E.v.H. performed bioinformatics analysis; A.-P.J. and M.T. performed cryo-EM-based molecular modeling; G.S. and B.J.G. performed molecular dynamics calculations; and A.J.R. performed subtilisin–MT TIRFM assays.

Corresponding authors

Correspondence to Carolyn A Moores or Anna Akhmanova.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 C-terminal domains of CAMSAP3 do not confer MT minus-end specificity.

(a) Schematic of the CAMSAP3 domain organization and the constructs used.

(b,c) TIRFM images and quantification of the binding of GFP-CAMSAP3 fragments to GMPCPP-stabilized MTs and dynamic GDP-MTs; values for GFP- CAMSAP1-D2 quantified in the same way are shown for comparison. The intensity is normalized to the average intensity of MBD on GMPCPP MTs. Scale bar, 2 μm.

(d) Sequence alignment of the linker region located upstream of the third coiled-coil in vertebrate CAMSAP1. Negatively and positively charged residues are highlighted in purple and cyan, respectively.

See also Supplementary Table 2.

Supplementary Figure 2 The CKK is a highly conserved MT minus-end-tracking domain.

(a) The presence of CKK in extant eukaryotic lineages and its evolutionary history since LECA. The CKK domain can be found in four out of five eukaryotic supergroups: Eukaryotic supergroups are indicated by colored branches (Opisthokonta: purple, Amoebozoa: blue, Stramenopila-Alveolata-Rhizaria (SAR): red, Archaeplastida: green, Excavata: brown). Species tree depicting all species comprising our eukaryotic proteome database and the presences of the CKK domain as well as its inferred losses. The presence of CKK in LECA and indicated subsequent loss events were inferred according to Dollo parsimony, which allows for only a single invention. Asterisk: sequence derived from NCBI. Note that in the oomycetes, the loss of CKK domain seems relatively recent, because Phytophthora infestans and Albugo laibachii contain CKK, but downy mildew Hyaloperonospora parasitica lacks it.

(b) Multiple sequence alignment of the CKK domain in a subset of the species in (a). The supergroups to which the species encoding these sequences belong are indicated by the name colors as in (a). The numbering of amino acids is based on human CAMSAP1 sequence. The colored asterisks indicate the amino acids, which, when mutated to alanine, alter the CAMSAP1mini signal at MT minus ends compared to wild type protein; red asterisk, signal reduced to less than 25%; black asterisk, signal reduced to 25-75%; cyan asterisk, signal increased above 100%.

Supplementary Figure 3 The X-ray crystal structure of the mouse CAMSAP3 CKK domain.

(a) Two views 180° apart of the ribbon representation of the M. musculus CAMSAP3 CKK domain crystal structure. Key loops as well as the N- and C-termini of the domain are labeled.

(b) The M. musculus CAMSAP3 CKK sequence, colored according to side chain property, is shown, with overlying secondary structure based on our crystal structure. The boxed region indicates the construct used for X-ray crystallography. Secondary structure estimations deduced by X-ray crystallography are indicated above, color coded consistent with the scheme in panel a, with unsolved regions indicated with dashed lines.

Supplementary Figure 4 Evaluation of the resolution of CAMSAP–CKK MT reconstructions, conservation of the CKK binding site in CAMSAP1 and comparison of the CKK binding site with those of other MAPs and MT motors.

(a) Protofilament skew for 16 representative MTs from each dataset is depicted by plotting the average rotation angle around the MT axis (PHI) change per dimer moving axially towards the MT plus-end. CAMSAP CKK-decorated MTs are compared to control kinesin decorated paclitaxel-stabilized MTs (datasets from Atherton, J. et al., eLife 3, e03680,2014). that show no skew for 13 pfs and a left-handed (negative) skew for 14pfs. PHI angles were determined in Frealign (Grigorieff, N., Journal of structural biology 157, 117-125, 2007). CAMSAP CKK decorated 13pf MTs show right-handed (positive) skew, while control 13pf kinesin decorated MTs show little or no skew. In contrast, control 14pf kinesin decorated MTs show left-handed (negative) skew as expected (Chretien, D. & Wade, R.H., Biology of the cell / under the auspices of the European Cell Biology Organization 71, 161-174, 1991). n=16 MTs per condition. Data represent mean ± SD. CAMSAP-3 13pf vs CAMSAP-1 13pf not significant, one-way ANOVA with Tukey’s multiple comparison test. CAMSAP-3 13pf vs kinesin-3 13pf, p<0.0001. CAMSAP-3 13pf vs kinesin-1 13pf, p<0.0001. CAMSAP-1 13pf vs kinesin-3 13pf, p<0.0001. CAMSAP-1 13pf vs kinesin-1 13pf, p<0.0001.

(b) The asymmetric reconstruction of the CAMSAP1-CKK decorated 13pf taxol-stabilized MT low-pass filtered to 15Å resolution shows extra densities (green) every 8 nm corresponding to the CAMSAP1-CKK domain, which are absent at the seam (arrow).

(c, d) Fourier shell correlation (FSC) curves utilizing the gold-standard noise substitution method (Chen, S. et al., Ultramicroscopy 135, 24-35, 2013) give an overall resolution estimate for the (c) CAMSAP3-CKK-MT reconstruction of 5.3Å and (d) CAMSAP1-CKK-MT reconstruction of 8Å.

(e) The averaged reconstruction of the CAMSAP1-CKK domain viewed from the MT surface contacting two β-tubulins and two α-tubulins at the intra-dimer, inter-protofilament interface. The CAMSAP-1 CKK is colored as in the CAMSAP3-CKK. α-tubulin is shown in light grey and β-tubulin is shown in dark grey. Above, schematic.

(f-i) Schematic depiction of the CKK binding site compared to other known MT binding partners, and structural models of (g) CAMSAP-CKK and EB1-CH domain (based on PDB ID 3JAK, Zhang et al., E. Cell 162, 849-859, 2015) (h) CAMSAP-CKK and DCX-DC domain (same binding site as EB, based on PDB ID 2XRP, Fourniol, F.J. et al., The Journal of cell biology 191, 463-470, 2010) and (i) CAMSAP-CKKs and kinesin-1 motor domain (based on PDB ID 4UY0, Atherton, J. et al., eLife 3, e03680,2014, note steric overlap).

See also Supplementary Table 2.

Supplementary Figure 5 Details of the CKK MT-binding site by ssNMR and cryo-EM.

(a) Zooms of well-resolved regions in the solid-state 13C-13C (PDSD, left and middle panels) and NCA (right panel) ssNMR spectra of [15N,13C,] labeled CKK bound to MTs. Crosses indicate expected signals based on solution-state chemical shifts of free CKK in solution. Signals for which a good match is found between expected and observed chemicals shifts are indicated in blue, while the absence of a corresponding signal is indicated in red. Brown crosses correspond to expected signals in overlapping regions.

(b) Full list of affected (red) and unaffected (blue) residues based on the NMR data.

(c, d) View from the MT plus-end (c) or minus-end (d) of the MT-bound CKK, reporting on ssNMR data obtained on [13C,15N] labeled CKK decorated MTs relative to NMR results on free CKK. The CKK is shown as a color-coded surface representation whilst a ribbon representation of the MT (light grey for α-tubulin and dark grey for β-tubulin) is shown within its respective grey transparent solid cryo-EM density. CKK residues that underwent significant chemical-shift/intensity changes are colored in red (affected) and those that experienced no change are indicated in blue (unaffected). White residues were not analyzed due to signal overlap.

(e) A view from the plus end of the MT, showing details of the tubulin contacts of the CKK N-terminus (red), helix α1, loop1 (magenta) and loop7 (yellow), colored as in Fig. 2d. The experimental density is shown as transparent solid, with tubulin shown as grey ribbons fitted into the experimental density.

(f) A view from the minus end of the MT, showing details of the tubulin contacts of the CKK N-terminus (red), loop7 (yellow), the C-terminus (blue) loop3 (orange) and the position of loop8 (cyan).

Supplementary Figure 6 Characterization of the CKK MT-binding site; energetics and electrostatic contributions.

(a) CAMSAP3 residue-wise variation in solvent accessible surface area (top, black bars) and energetic contributions to MT binding (bottom, green bars). Selected CKK residues showing major solvent accessibility changes and energetic contributions are labelled in green, with corresponding interacting tubulin residues labelled in light grey for α-tubulin and dark grey for β-tubulin. Red stars indicate the location of residues with ssNMR signal shifts. Energetic contributions were determined from molecular dynamics simulations using the Amber MMGBSA routine. Data represent mean ± SD. Solvent accessibility was determined with Bio3D (Grant, B.J. et al., Bioinformatics 22, 2695-2696, 2006, see Methods).

(b) Surface representation of the MT-CKK interface on tubulin colored by electrostatic potential, with the CKK binding site indicated by a green dashed outline. The surface was calculated with the APBS (Baker, N.A. et al., PNAS 98, 10037-10041, 2001) and pdb2pqr (Dolinsky, T.J. et al., Nucleic acids research 35, W522-525, 2007).

(c) The corresponding front view of the surface-represented CKK (left) colored in the same way as panel b, while a 180° rotation of this view illustrating the tubulin-facing surface of the CKK is shown on the right.

(d) MT pelleting assay, in which the CAMSAP3 CKK and MTs were mixed in equimolar ratios at different salt concentrations in BRB35 buffer. As controls, the CKK domain and MTs were processed alone. The supernatants (S) and pellets (P) were separated on a 15 % SDS-PAGE and stained with Coomassie.

(e) Tubulin-facing surface representation of CAMSAP3 CKK surface showing the residue conservation between the M. musculus, C. elegans, D. melanogaster CKK domains. Dark green, invariant residues; light green, similar residues; white, non-conserved residues. Equivalent view to right hand image in panel (c) is shown.

Supplementary Figure 7 Validation of CKK–MT contact sites by using CKK mutants.

(a) CD spectra (left) and thermal unfolding profiles (right) of CAMSAP3 CKK variants. The spectra were recorded at 5 °C in PBS at a protein concentration of 0.25 mg/ml. The thermal unfolding profiles were recorded at 206 nm.

(b,c) TIRFM images and quantification of GFP-CAMSAP3 CKK and N1130A mutant binding to GMPCPP-stabilized MTs. The intensity is normalized to the average minus-end intensity of WT. Similar to CAMSAP1 N1492A mutant, CAMSAP3 N1130A mutant shows increased MT lattice binding. n=80 MTs.

(d-f) TIRFM images and quantifications of the binding of GFP-CAMSAP1mini N1492A and N1492T mutants to the minus ends and lattice of dynamic MTs at different concentrations. Both mutants show minus-end specificity at lower concentrations (see Fig. 4a,d for images at 50 nM). The intensity is normalized to the average minus-end intensity of wild type. n=30-50 MTs. The data for N1492A and N1492T mutants at 50 nM was replotted from Fig. 4d.

(g,h) TIRFM images and quantification of binding of GFP-CAMSAP1mini and mutants to the minus ends and lattice of dynamic MTs. The intensity is normalized to the average minus-end intensity of wild type. n=30 MTs.

Scale bars, 1 μm. Data represent mean ± SD.

See also Supplementary Table 2.

Supplementary Figure 8 Reconstruction of the CAMSAP1 N1492A CKK mutant and analysis of the effect of CKK on MT polymerization.

(a) The asymmetric reconstruction of the CAMSAP1-N1492A CKK decorated 13pf paclitaxel-stabilized MT low-pass filtered to 15Å resolution shows extra densities (green) every 8nm corresponding to the CAMSAP1-N1492A CKK domain, which are absent at the seam similar to wild type (arrow).

(b) FSC curves utilizing the gold-standard noise substitution method (Chen, S. et al., Ultramicroscopy 135, 24-35, 2013) give an overall resolution estimate for the CAMSAP1-N1492A CKK-MT reconstruction of 4.8Å.

(c) The averaged reconstruction of the CAMSAP1- N1492A CKK domain viewed from the MT surface contacting two β-tubulins and two α-tubulins at the intra-dimer, inter-protofilament interface. The CAMSAP1-N1492A CKK is colored as previously. α-tubulin is shown in light grey and β-tubulin is shown in dark grey. Cryo-EM density is shown as transparent light blue solid. Above, schematic.

(d) View from the plus end of the CAMSAP1-N1492A CKK decorated 13pf MT, showing details of the tubulin contacts of the N-terminus (red), helix α1, loop1 (magenta) and loop 7 (yellow), colored as in Supplementary Fig. 5e. The position of N1492A (brown) is also indicated (arrowhead). The experimental density is shown as blue transparent solid, with tubulin shown as grey ribbons fitted into the experimental density.

(e) Protofilament skew for representative MTs from each dataset is depicted as in Supplementary Figure 4a. The reduced skew produced by CAMSAP1-N1492A CKK-decorated MTs are compared to wild type CKK MTs and control kinesin decorated paclitaxel-stabilized MTs (datasets from Atherton, J. et al., eLife 3, e03680,2014). For CAMSAP1 CKK, n=16, for kinesin1 decorated MTs, n=16; for CAMSAP1-N1492A CKK-decorated MTs, n=15. Data represent mean ± SD. 1-way ANOVA, CAMSAP1 CKK N1492A vs CAMSAP1 CKK wild type, p<0.001;CAMSAP1 CKK N1492A vs kinesin-1 13pf p<0.05.

(f) Copolymerisation of tubulin with CAMSAP1 CKK domain yields few MTs but produces clumps of heterogeneous tubulin oligomers (left), while copolymerisation with CAMSAP-N1492A yields mainly MTs (right). Representative cryo-EM images of the products of each polymerisation reaction are shown.

See also Supplementary Table 2.

Supplementary Figure 9 Determination of MT polarity in cryo-tomograms.

(a) The moire pattern from successive raw images in a representative tilt series, and corresponding Fourier filtered images. The tilt method (Chretien, D. et al., Structure 4, 1031-1040, 1996) uses the position of the tilt axis and shift with tilt of the fringes (2 and 3) in the moiré pattern of 14pf MTs to identify MT polarity. The plus (+) end is oriented towards the top of the page.

(b) The moiré pattern of a subset of Fourier filtered MT projections also contain an arrowhead pattern, which arises from pf skew, which can be used to corroborate MT polarity (Chretien, D. et al., Structure 4, 1031-1040, 1996). In 14pf MTs, the arrowheads point towards the minus end; black arrowheads indicate the position of the arrowhead moire patterns on the MTs and thus demonstrate that the MTs are oriented with their plus ends towards the top of the image.

(c) 14 pf MT minus end model built from known tubulin dimer structures (PDB numbers indicated).

(d) Change in lateral pf separation compared to lattice was measured in transverse sections of the MT lattice and ends and plotted as frequency histogram (% of total observations). At both minus and plus ends, most pfs remain laterally closely associated despite longitudinal curvature (0 or +1 nm separation), consistent with a sheet-like structure, and in contrast to the lateral separation of the end model where pfs instead splay apart. Inset; a schematic of a transverse section through the MT lattice and an MT end indicating pf lateral separation distance in the lattice (L) and in the longitudinally curved end region (E).

(e) A scheme illustrating the curved region of a MT end in 2D. The measured axial length of the curved region is the distance between the two red dashed lines.

(f) Gallery of 2D projections of exemplar GMPCPP MT ends with or without 1.5 mg/ml CAMSAP3 CKK domain. Note similar end structures with moderate longitudinal curvature, but also the much higher background in the presence of excess CKK protein.

(g) 2D quantification of the axial length of curved regions of GMPCPP MT ends (as shown in panel e) in the presence or absence of CAMSAP3 CKK domain. No significant difference is observed with added CKK domain, two-tailed Mann-Whitney U test. For GMPCPP-MT ends, n=77 MT ends; for CKK-GMPCPP-MT ends, n=63 MT ends. Data represent mean ± SD.

(h) A histogram of axial length of curved regions of GMPCPP MT ends shows similar length distributions in the presence or absence of CAMSAP3 CKK domain. In both cases, the most common bin center of end lengths is 20 nm (bin width 10 nm).

See also Supplementary Table 2.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Note (PDF 3645 kb)

Life Sciences Reporting Summary (PDF 128 kb)

Supplementary Data Set 1

Full-length sequences of CKK domain-containing proteins derived from a reference proteome set of 99 eukaryotic species. (ZIP 84 kb)

Supplementary Table 2

Source data (XLSX 191 kb)

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Atherton, J., Jiang, K., Stangier, M. et al. A structural model for microtubule minus-end recognition and protection by CAMSAP proteins. Nat Struct Mol Biol 24, 931–943 (2017). https://doi.org/10.1038/nsmb.3483

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