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A chemical genetics approach to examine the functions of AAA proteins

Abstract

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1—AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.

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Fig. 1: Engineering biochemically silent mutations in the ATP-binding site of katanin.
Fig. 2: Using RADD to analyze the binding of triazolopyridine-based compounds to katanin.
Fig. 3: Design of an allele-specific covalent inhibitor of katanin.
Fig. 4: Probing katanin function using a covalent inhibitor and sensitized allele pair.
Fig. 5: Allele-specific inhibition of the AAA proteins VPS4B and FIGL1.
Fig. 6: An approach for developing allele-specific covalent inhibitors for proteins in the AAA family.

Data availability

The structure of VPS4B-D135C bound to ASPIR-2 has been deposited in the Protein Data Bank (PDB) under accession code PDB 7L9X. Data generated or analyzed during this study are included in this published Article (and its Supplementary Information files). Source data are provided with this paper.

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Acknowledgements

We thank J. Ross (UMass Amherst) for providing the H. sapiens katanin-p60 plasmid. T.M.K. is grateful to the NIH (GM130234) and Starr Cancer Consortium (I12-0055) for supporting this research. T.C. was supported in part by the 2018 Kestenbaum Award for Neurodegeneration. N.H.J. was supported in part by the National Science Foundation Graduate Research Fellowship Program (2017242069) and the NIH T32 GM115327 Chemistry-Biology Interface Training Grant to the Tri-Institutional PhD Program in Chemical Biology. M.J.G. was supported in part by an NCI T32 Training Grant (5 T32 CA 9673-40). We are grateful to the High Throughput and Spectroscopy Resource Center at The Rockefeller University for instrument use. We also thank Michael Oldham and Jue Chen (The Rockefeller University) and Deena Oren (Structural Biology Resource Center at The Rockefeller University) for equipment use and for engaging in helpful discussions regarding crystallography experiments. This research used the 17-ID-1 beamline of the National Synchrotron Light Source II, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under contract no. DE-SC0012704.

Author information

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Authors

Contributions

T.C., N.H.J. and T.M.K. conceived the project and designed experiments. T.C., N.H.J., M.J.G. and R.P. carried out protein biochemistry. T.C. and N.H.J. synthesized compounds. T.C. completed the computational docking analysis. T.C., N.H.J. and M.J.G. performed assays and analyzed data. N.H.J. engineered cell lines and acquired and analyzed cell imaging data. M.J.G. performed structural biology experiments. T.M.K. supervised the research. T.C., N.H.J. and T.M.K. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Tarun M. Kapoor.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks Petra Wendler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Inês Chen and Florian Ullrich were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Characterizing the binding mode of triazolopyridine-based compounds to katanin.

(a-b) DSF traces (a) and changes in melting temperatures (∆Tm, b) for kata-AAA-WT in the presence of compounds 1-4 (500 µM, n = 2). As a reference, the corresponding trace for control is shown (5% DMSO, dashed line, data in Fig. 1d). (c) Four computational docking models for compound 1 (purple and blue, stick representation) bound to the katanin nucleotide-binding site (gray, ribbon representation). Variability hotspot residues are shown (stick representation, color-coded as in Fig. 1a). Other key amino acids in the katanin ATP-binding site are also shown (gray, stick representation). Pose 1 corresponds to the one in Fig. 2e. (d) Isothermal Titration Calorimetry (ITC)-based analyses of kata-AAA-WT in the presence of compounds 1-3. Raw injection heats are shown for titrations of compounds 1-3 against kata-AAA-WT (left panels), or compound titrations into buffer (right panels). Compound 4 could not be analyzed under similar conditions due to limited solubility. (e) Integrated data points and fitted binding curves used to determine Kd values. Data for graphs in (a-b) and (d-e) are available as source data.

Source data

Extended Data Fig. 2 Engineering the active site of katanin to obtain an allele sensitized to covalent inhibitors.

a, Primary sequence of the katanin N-loop motif, which contains D210. b, Partial sequence alignment of AAA proteins showing the residues in the N-loop motif (D210 is indicated by the arrow, alignment generated using Clustal Omega). c, Schematic showing the AAA domain (light gray box, not to scale), and the first and last residues of the ATPase active human katanin construct. The position of the D210C mutation is indicated by the red bar. d, Differential scanning fluorimetry of katanin-WT and katanin-D210C in the absence and presence of ADP (1 mM) (n = 2 independent experiments). One representative experiment is shown. e, SDS–PAGE gels of purified recombinant L211C and T250C X. laevis katanin mutant constructs (Coomassie blue staining). Data for the graph in (d) and the unmodified gel picture for (e) are available as source data.

Source data

Extended Data Fig. 3 Characterization of kata-WT and kata-D210C cell lines and additional analyses of ASPIR-1.

a, Full blots for Fig. 4a. Doxycycline (10 ng/mL, 14 hours) was used to induce expression and blot was stained for katanin (left panel) and GAPDH (right panel) as a loading control. The positions of the bands expected for the EGFP-katanin construct and the endogenous katanin (black and gray arrows, respectively), or GAPDH (black dotted arrow) are indicated. A representative blot is shown (n = 2 independent cell cultures per cell line). (b-c) Maximum intensity confocal projections show EGFP distribution in interphase (b) and dividing (c) HeLa cells expressing WT or D210C EGFP-katanin. Representative images are shown (n = 2 independent experiments, 10 images acquired per experiment). d, Kata-WT cells were incubated with different concentrations of ASPIR-1 for 24 h and viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay. Data are mean ± s.d., n = 3 independent experiments. e, Microtubule organization in fixed kata-WT cells treated for 4 hours with ASPIR-1 (1.25 µM) or control (DMSO, 0.1%) and stained for α-tubulin. Representative images, identically contrasted maximum intensity projections are shown (n = 3 independent experiments, 10 images acquired per condition per experiment, scale bar =10 µm). The uncropped blots for (a) and data for the graph in (d) are available as source data.

Source data

Extended Data Fig. 4 Effect of the inhibitor-sensitizing Asp-to-Cys mutation on the AAA proteins VPS4B and FIGL1.

a, SDS–PAGE analysis of purified recombinant human wild type (WT) and mutant (D135C) HS-VPS4B constructs, and wild type and mutant (D402C) FIGL1 constructs (Coomassie blue staining). b, Percentage steady-state ATPase activity of FIGL1 and HS-VPS4B (WT) in the presence of ASPIR-1 (5 µM, 1 mM ATP, 30 min incubation; data represent mean ± s.d., n = 3 independent experiments). (c-d) ATP concentration dependence of the steady-state activity of WT and D135C HS-VPS4B (c), and WT and D402C FIGL1 (d) analyzed using an NADH-coupled assay. Rates were fit to the Michaelis–Menten equation for cooperative enzymes (mean ± range, n = 2 independent experiments for HS-VPS4B-WT and HS-VPS4B-D135C; mean ± s.d., n = 5 independent experiments for FIGL1-WT, n = 7 independent experiments for FIGL1-D402C). Kinetic parameters were determined: kcat = 1.7 ± 0.1 s-1, K1/2 = 0.12 ± 0.07 mM for HS-VPS4B-WT; kcat = 0.5 ± 0.1 s-1, K1/2 = 0.15 ± 0.01 mM for HS-VPS4B-D135C; kcat = 3.4 ± 0.4 s-1, K1/2 = 0.2 ± 0.1 mM for FIGL1-WT; kcat = 2.3 ± 0.5 s-1, K1/2 = 0.3 ± 0.1 mM for FIGL1-D402C. (e-f) Differential scanning fluorimetry of WT and D135C HS-VPS4B (e) and WT and D402C FIGL1 (f) in the absence and presence of ADP (1 mM) (5% DMSO for both conditions). One representative experiment is shown (n = 2 independent experiments). The unmodified gel images for (a) and data for the graphs in (b-f) are available as source data.

Source data

Extended Data Fig. 5 Inhibition of VPS4B-D135C by ASPIR-2.

a, SDS–PAGE analysis of purified recombinant human wild type (WT) and mutant (D135C) VPS4B (tagless), and VTA1 constructs (Coomassie blue staining). b, ATP-concentration dependence of the steady-state activity of VPS4B-WT and VPS4B-D135C in the presence of 2-fold excess VTA1, analyzed using an NADH-coupled assay. Rates were fit to the Michaelis–Menten equation for cooperative enzymes (mean±range, n = 2 independent experiments). c, Chemical structure of ASPIR-2, the analog used for x-ray crystallography studies. d, Time-dependent inhibition of the ATPase activity of VPS4B-D135C by ASPIR-2. Graph shows percentage residual ATPase activity (mean ± range, n = 2 independent experiments). e, Concentration-dependent inhibition of the VTA1-stimulated, steady-state ATPase activity of WT and D135C VPS4B after 30 min incubation with ASPIR-2 (1 mM ATP; data represent mean ± range, n = 2 independent experiments). f, 2Fo-Fc electron density map of the crystal structure of VPS4B-D135C bound to ASPIR-2, contoured at 2.0 σ. g, Overlay of the structure of VPS4B-D135C in complex with ASPIR-2 with the RADD model for compound 1 bound to katanin, at the nucleotide-binding site (ASPIR-2: purple and blue, compound 1: pink and blue, stick representation; VPS4B-D135C: gray, katanin: white, ribbon representation; VPS4B residue Cys-135 is also shown). The unmodified gel image for (a) and data for the graphs in (b) and (d-e) are available as source data.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–2 and Notes 1–2.

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Supplementary Data 1

List of numbered compounds with structures and IUPAC names.

Source data

Source Data Fig. 1

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Cupido, T., Jones, N.H., Grasso, M.J. et al. A chemical genetics approach to examine the functions of AAA proteins. Nat Struct Mol Biol 28, 388–397 (2021). https://doi.org/10.1038/s41594-021-00575-9

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