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An allosteric network in spastin couples multiple activities required for microtubule severing

Nature Structural & Molecular Biology volume 26, pages 671–678 (2019)Cite this article

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An Author Correction to this article was published on 19 March 2020

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Abstract

The AAA+ ATPase spastin remodels microtubule arrays through severing and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.

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Fig. 1: The architecture of the substrate-bound spastin hexamer in a split lock-washer conformation.
Fig. 2: A double-helical staircase of pore loops surrounds the substrate and is coupled by a third pore loop spiral to the ATP binding site.
Fig. 3: A pore loop network couples nucleotide binding to substrate engagement and oligomerization.
Fig. 4: Mutations in the three pore loops impair ATPase and microtubule severing.
Fig. 5: An allosteric network mutated in HSP couples substrate binding to oligomerization and ATP hydrolysis.
Fig. 6: Structure-based insight into the mechanism of action of human spastin HSP disease mutations.

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Data availability

Electron microscopy map and the top scoring model of five atomic models obtained from an EM multi-model pipeline have been deposited at the Electron Microscopy Data Bank and Protein Data Bank under accession numbers EMD-20226 and PDB 6P07, respectively. All data used in this study are available from the corresponding authors upon reasonable request.

Change history

  • 19 March 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank J.C. Ducom at The Scripps Research Institute High Performance Computing for computational support and B. Anderson at The Scripps Research Institute electron microscopy facility for microscope support. We thank M. Herzik and A. Hernandes for help with atomic modeling, E. Szczesna for help with microtubule severing assays, G. Piszcek from the Biophysics Core of the National Heart, Lung and Blood Institute (NHLBI) for help with AUC experiments, S. Chowdhury, C. Puchades and M. Wu for helpful discussion. C.R.S. was supported by a National Science Foundation predoctoral fellowship. G.C.L. was supported as a Searle Scholar, a Pew Scholar, an Amgen Young Investigator and by the National Institutes of Health (NIH) grant no. DP2EB020402. Computational analyses of EM data were performed using shared instrumentation funded by NIH grant no. S10OD021634 to G.C.L. A.R.M. was supported by the intramural programs of the National Institute of Neurological Disorders and Stroke (NINDS) and the NHLBI.

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Authors and Affiliations

  1. The Scripps Research Institute, La Jolla, CA, USA

    Colby R. Sandate & Gabriel C. Lander

  2. Cell Biology and Biophysics Unit, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA

    Agnieszka Szyk, Elena A. Zehr & Antonina Roll-Mecak

  3. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, MD, USA

    Antonina Roll-Mecak

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Contributions

C.R.S. froze EM grids, collected and processed EM data and built atomic models. A.S. purified all proteins, performed AUC and ATPase assays. E.A.Z. performed severing assays. C.R.S., G.C.L. and A.R.M. interpreted structural models and wrote the manuscript.

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Correspondence to Gabriel C. Lander or Antonina Roll-Mecak.

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Supplementary Figure 1 Analytical ultracentrifugation and ATPase assays.

(a) AUC experimental distribution for the spastin Walker B mutant in the presence of ATP (Methods). The expected molecular weight of the spastin hexamer is 320 kDa. (b) Spastin ATPase stimulation by poly-glutamate (0.75–5 KDa) used for our structural determination; n=4; lines indicate mean and S.D. (c) AUC experimental distribution for the spastin Walker B mutant in the presence of ATP and Atto-488 labeled poly-glutamate peptide showing co-migration of the spastin hexamer and peptide. AUC experimental distribution for the spastin Walker B mutant alone at 6μM in the presence of ATP (top panel), Atto488-labeled VGSEEEEEEEEEE peptide at 16.7 μM in the presence of ATP (middle panel), spastin Walker B mutant at 10 μM (1.67 μM for the hexamer) and Atto-labeled peptide at 16.7 μM in the presence of ATP (lower panel).

Supplementary Figure 2 Structural comparison of the final reconstructions of the spastin hexamer with poly-Glu substrate and the 3.8 Å resolution map of the spastin hexamer obtained without the addition of substrate.

(a) EM density of the earlier reconstruction (yellow) adjacent to our final reconstruction with poly-Glu added (grey). The sample preparation for this structure did not include incubation in the presence of substrate. Despite this, an unknown density was found docked within the pore of the hexamer. Cryo-EM data pertaining to this map was processed in a similar fashion to our 3.2 Å map, except using Relion 2.1 instead of cryoSPARC for the final 3D reconstruction. Incubation of our sample with poly-Glu increased the number of intact hexameric particles used in the reconstruction and allowed us to acquire a map at higher resolution. (b) Comparison of raw micrograph images selected from both datasets. (c) Comparison of 2D classes selected from both datasets. (d) Comparison of substrate density for the final reconstruction (grey, poly-Glu substrate is light green) and unknown substrate density (yellow, unknown density is purple). (e) Euler distribution plot for the final reconstruction with the poly-Glu substrate. More populated views are shown in red.

Supplementary Figure 3 Image processing of cryo-EM data and local and global resolution of the final cryo-EM map.

(a) Flowchart for image processing of cryo-EM data. (b) Local resolution estimation of the final cryo-EM map using BSOFT85. (c) Gold standard Fourier shell correlation versus spatial frequency plot of final map refinement in cryoSPARC74. Blue horizontal line depicts the gold standard FSC (GSFSC) value equal to 0.143. (d) Multi-model validation79: per-residue Cα RMSD values were calculated from the top ten refined atomic models and plotted on the histogram with the mean per-residue value denoted by a black vertical bar. Inset is the atomic model in worm representation colored by per-residue Cα RMSD, as in the histogram.

Supplementary Figure 4 EM map quality at selected secondary structural elements and EM map and atomic model of the nucleotide binding pocket of each protomer.

(a) Quality of the EM map at selected secondary structural elements. The entirety of protomer C is shown in ribbon representation and colored according to Fig. 1 with the corresponding EM density shown as a transparent surface. Selected structural elements are labelled and shown in stick representation with EM density depicted in grey mesh. (b) EM map and atomic model of the nucleotide binding pocket of each protomer. Sidechains and nucleotides are depicted in stick representation, while the backbone is in ribbon and the EM map is shown as grey mesh. Each chain is colored and labeled according to its respective protomer (see Fig. 1b). For the ATP in protomers A through E, clear distinctive density was observed consistent with a coordinating magnesium ion. The nucleotide density for protomer F is less well resolved and may contain a mixture of ADP and ATP states.

Supplementary Figure 5 Structural alignment of spastin protomers within the hexamer and comparison to apo spastin monomer.

(a) Structural alignment of each protomer using Chimera’s Match Maker86 with a Cα-RMSD of 0.685 angstroms. (b) Superposition of the apo spastin monomer (light grey; PDB: 3B9P6) and the ATP-bound protomer C from our hexameric spastin structure (dark grey) with pore loops 1, 2 and 3 highlighted in blue, yellow and magenta, respectively. The pore loops in the apo structure are shown in lighter hues of the same colors. The NBD undergoes large structural rearrangements upon ATP and peptide substrate binding, resulting in repositioning of pore loop 1, a disorder-to-order transition for pore loop 2 and ~ 1 Å movement of pore loop 3. Additionally, the HBD of the ATP and substrate-bound protomer undergoes a rotation of 9 degrees away from the NBD relative to the apo protomer.

Supplementary Figure 6 Oligomerization interactions between spastin protomers.

(a) The linker (purple) and helix α1 (blue) participate in oligomerization interactions. (b) The C-terminus of spastin is stabilized in the oligomer and together with helix α11 (orange) forms a belt around the hexamer. (c) Invariant Y753 is part of the oligomerization interface with the α10-α11 loop. Protomer D is depicted in blue, protomer E in green. The cryo-EM density is shown as a semi-transparent surface. (d) R601 is within H-bonding distance to the polyglutamate substrate and S599 of the adjacent lower protomer, likely coupling oligomerization to substrate engagement. Protomers colored as in Fig. 1.

Supplementary Figure 7 Modeled substrate within the cryo-EM density.

(a). Poly-glutamate substrate shown in two opposing orientations with the EM density shown as a mesh. (b) Stereo view of the poly-glutamate fit into the EM density.

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Sandate, C.R., Szyk, A., Zehr, E.A. et al. An allosteric network in spastin couples multiple activities required for microtubule severing. Nat Struct Mol Biol 26, 671–678 (2019). https://doi.org/10.1038/s41594-019-0257-3

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  • DOI: https://doi.org/10.1038/s41594-019-0257-3

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