Figure 6. Analysis of the mechanism of dynein inhibition by dynapyrazole-A (compound 8).

(A) Basal ATPase activity of GFP-dynein in the solvent control (2% DMSO, n = 8) and in the presence of 8 (40µM, n = 4) and ciliobrevin D (40 µM, n = 4). (B) SDS-PAGE analysis (Coomassie blue stain) of His-dynein 1, ~0.5 µg protein loaded. Mass spectrometry showed that the impurity (~15%, triangles) is likely to be a fragment of dynein (Figure 6—figure supplement 1) (C) Basal ATPase activity of His-dynein in the solvent control (2% DMSO, n = 11) and in the presence of 8 (40µM, n = 11) and ciliobrevin D (40 µM, n = 5). (D) Microtubule-stimulated ATPase activity of His-dynein 1 across a range of microtubule concentrations in the solvent control (2% DMSO) or in the presence of 30 µM 8 (2.5 µM microtubules, n = 5; 8 µM microtubules, n = 4; 8 µM microtubules, 30 µM 8, n = 3; 15 µM microtubules, 2% DMSO, n = 4; 15 µM microtubules, 30 µM 8, n = 3) (E) Dose-dependent inhibition of microtubule-stimulated His-dynein 1 ATPase activity by 8 (2.5 µM microtubules, IC₅₀: 6.2 ± 1.6 µM, n = 3). (F) SDS-PAGE analysis (Coomassie blue stain) of dynein following irradiation with ultraviolet light at 365 nm. The components included in the photocleavage reaction loaded into each lane are indicated above the lane. Arrowheads indicate dynein photocleavage products. (G) Analysis of gel band intensity for photocleavage reactions. Values are mean + S.D., n = 3. (H) Gel filtration traces (Superose 6) for His-dynein 1 wild-type and AAA3 Walker A mutant. Peak elution volumes are 12.2, and 12.4 mL, respectively. V_o, void volume. (I) SDS-PAGE analysis (Coomassie blue stain) of Walker A mutant His-dynein 1 protein, ~0.5 µg protein loaded. Mass spectrometry data confirming the presence of the K2601A mutation in this construct is shown in Figure 6—figure supplement 4. (J) Basal and microtubule-stimulated ATPase activity of the AAA3 Walker A-mutant His-dynein 1 in the solvent control (2% DMSO) and in the presence of 8 (30µM). (K) Inhibition of the basal ATPase activity of the AAA3 Walker A-mutant His-dynein 1 by 8 (IC₅₀: 5.5 ± 1.6 µM, n = 5) and ciliobrevin D (IC₅₀: 38.4 ± 6.3 µM, n = 3). IC₅₀ values reported reflect the mean (±S.D.) of separate IC₅₀ values obtained from independent dose-response analyses. For (E) and (K), data were fit to a four-parameter sigmoidal dose-response curve in PRISM and fits were constrained such the value at saturating compound >0. All ATPase assays were performed at 1 mM MgATP and 2% DMSO. All data presented are mean ± S.D. of n ≥ 3 data points, except in K, where replicate numbers for individual datapoints were as follows. 8: 80 µM-2, 40 µM-5, 20 µM-5, 10 µM-5, 5 µM-5, 2.5 µM-5, 1.3 µM-5, 0.6 µM-2. Ciliobrevin D: 80 µM-2, 40 µM-3, 20 µM-3, 10 µM-3, 5 µM-3, 2.5 µM-3.

DOI: http://dx.doi.org/10.7554/eLife.25174.024

Figure 6—figure supplement 1. Mass spectrometry-based analysis of wild-type His-dynein 1.

Protein sample was run on an SDS-PAGE gel. The largest band at >350 KDa and a minor band (indicated with triangles in Figure 6B) were excised from a gel and analyzed. Peptides identified by mass spectrometry are indicated (green bars, schematic generated using Proteome Discoverer 1.4, Thermo Scientific). Common contaminants are excluded (e.g. trypsin, keratin). The minor band was also identified as His-dynein 1 and is likely a product of partial proteolysis.

DOI: http://dx.doi.org/10.7554/eLife.25174.025

Figure 6—figure supplement 2. Dose-dependent inhibition of microtubule-stimulated His-dynein 1 ATPase activity by ciliobrevin D (2.5 µM microtubules).

Data are presented as mean ± range, n ≥ 2.

DOI: http://dx.doi.org/10.7554/eLife.25174.026

Figure 6—figure supplement 3. Purification and testing of His-dynein 1 with Walker A mutation in AAA1.

Gel filtration trace (Superose 6) for His-dynein 1 with K1912A (AAA1 Walker A lysine to alanine), with volume at elution peak indicated. SDS-PAGE analysis (Coomassie stain) of this protein, ~0.5 µg protein loaded. V_o, void volume. The ATPase activity for this enzyme was measured as 0.05–0.1 s⁻¹ and was <2x above background hydrolysis in the absence of enzyme.

DOI: http://dx.doi.org/10.7554/eLife.25174.027

Figure 6—figure supplement 4. Mass spectrometry-based analysis of His-dynein 1 with Walker A mutation in AAA3.

Protein samples of wild-type and mutant (K2601A, AAA3 Walker A lysine to alanine) were run in separate lanes of an SDS-PAGE gel. Gel bands for each protein were excised, trypsinized. Peptides allowing for the differentiation of dynein 1 wild-type and mutant (all in the Walker A region) and also peptides that could serve as loading controls were targeted in a parallel reaction monitoring experiment (Peterson et al., 2012). Data were analyzed using Skyline (MacLean et al., 2010) combined with ProteomeDiscoverer 1.4 (Thermo Fisher) and Mascot (Matrix Science). For each protein, peptide counts for the peptides GPPGSGATMTLFSALR, GPPGSGK, and TMTLFSALR were divided by the sum of counts for all three peptides and this value is presented below. GPPGSGATMLFSALR peptide is expected in the AAA3 mutant, whereas GPPGSGK and TMTLFSALR are expected in the wild-type enzyme.

DOI: http://dx.doi.org/10.7554/eLife.25174.028

Figure 6—figure supplement 5. Sequence analysis of human cytoplasmic dynein isoforms 1 and 2.

Alignments were performed using the ClustalW algorithm for the following protein fragments: (A) human dynein 1 AAA1 (T1882-Y2193) and human dynein 1 AAA3 (T2571-L2911) (B) human dynein 1 AAA1 (T1882-Y2193) and human dynein 2 AAA1 (T1665-I1954). Residues in in black boxes are identical across isoforms. Red stars denote residues < 4 Å from the nucleotide (ADP-Vanadate) in AAA1 of human dynein 2 (PDB 4RH7). Some residues within this 4 Å shell are not included in this sequence alignment because they are located in adjacent AAA domains. Domain boundaries were chosen based on analysis of available crystal structures and sequence alignments. Uniprot accession numbers: dynein 1-Q14204 dynein 2-Q8NCM8.

DOI: http://dx.doi.org/10.7554/eLife.25174.029