Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect

Abstract

Microtubules, composed of αβ-tubulin heterodimers, have remained popular anticancer targets for decades. Six known binding sites on tubulin dimers have been identified thus far, with five sites on β-tubulin and only one site on α-tubulin, hinting that compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our x-ray crystallography study reveals that, in addition to binding to the vinblastine site, cevipabulin also binds to a new site on α-tubulin. We find that cevipabulin at this site pushes the αT5 loop outward, making the nonexchangeable GTP exchangeable, which reduces the stability of tubulin, leading to its destabilization and degradation. Our results confirm the existence of a new agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of antimicrotubule drugs targeting this novel site.

Fig. 1. Cevipabulin promotes tubulin dimer degradation.

(A) Label-free quantitative proteomic analysis of total proteins from HeLa cells treated with 1 μM cevipabulin for 6 hours. This graph presents fold changes of 1825 quantified proteins between cevipabulin and DMSO treatment groups versus the P value (t test; triplicate analysis). Three biological repetitions are performed. (B) Immunoblotting analysis of both α- and β-tubulin levels in HeLa, Hct116, H460, and SU-DHL-6 cells, which all are treated with indicated concentrations of cevipabulin for 16 hours. Results are representative of three independent experiments. (C) HeLa cells were treated with 1 μM cevipabulin for the indicated times, and then the α- and β-tubulin levels were detected by immunoblotting. Results are representative of two independent experiments. (D) HeLa and Hct116 cells were treated with indicated concentrations of cevipabulin for 16 hours, and then mRNA levels of both α-tubulin and β-tubulin were measured by quantitative-PCR. Data were shown as means ± SD of three independent experiments. (E) Cells were treated with or without MG132 (20 μM) for 1 hour before treated with different concentrations of cevipabulin for 16 hours. Protein levels of both α- and β-tubulin were detected by immunoblotting. Results are representative of two independent experiments. Cev, cevipabulin; Tub, tubulin.

Fig. 2. Crystal structure of the cevipabulin-tubulin complex.

(A) Chemical structure of cevipabulin. (B) Overall structure of the cevipabulin-tubulin complex. TTL is colored yellow, RB3 is green, α-tubulin is black, and β-tubulin is gray. Cevipabulin on β1-tubulin and α2-tubulin are all shown in spheres and colored yellow. (C and D) Electron densities of cevipabulins on (C) β1-tubulin or (D) α2-tubulin. The F_obs/F_calc omit map is colored light blue and contoured at 3δ. (E to G) Close-up view of vinblastine-site cevipabulin binding to (E and F) β1-tubulin or (G) α2-tubulin. GDP or GTP is shown in magenta sticks. Cevipabulin is shown in yellow sticks. Side chain of β1-Y224 or α2-Y224 is shown in gray sticks. (H) Interactions between the seventh-site cevipabulin and α2-tubulin. Color coding is the same as in (E). Residues from tubulin that form interactions with vinblastine-site cevipabulin are shown as sticks and labeled. Hydrogen bonds are drawn with red dashed lines. (I) Interactions between α2-tubulin and the seventh-site cevipabulin; Color coding is the same as in (F); residues from tubulin that form interactions with the seventh-site cevipabulin are shown as sticks and labeled. Hydrogen bonds are drawn with red dashed lines. Cev, cevipabulin.

Fig. 3. Measuring the binding stoichiometry of cevipabulin to tubulin.

(A) Indicated concentrations of cevipabulin were incubated with tubulin (20 μM) for 10 min, and then the bound cevipabulin was quantified by LC-MS/MS. This graph presented the molecular ratio of the cevipabulin:tubulin dimer. Data were shown as means ± SD of three independent experiments. (B) Indicated concentrations of cevipabulin were incubated with the eribulin-tubulin complex (20 μM) for 10 min, and then the bound cevipabulin and eribulin were quantified by LC-MS/MS. This graph presented the molecular ratio of the cevipabulin:tubulin or eribulin:tubulin dimer. Data were shown as means ± SD of three independent experiments. (C) Binding of cevipabulin to the seventh site of tubulin was determined with the microscale thermophoresis assay. Data points represent means ± SD of three technical replicates each. (D) Structure of the cevipabulin-eribulin-tubulin complex. TTL is colored yellow, RB3 is blue, α-tubulin is black, and β-tubulin is gray. Cevipabulin on β1-tubulin and α2-tubulin and eribulin on β2-tubulin were all shown in spheres and colored yellow. (E to G) Electron densities of cevipabulin on β1-tubulin (E) or α2-tubulin (F) and eribulin on β2-tubulin (G). The F_obs/F_calc omit map is colored light blue and contoured at 3δ. Cev, cevipabulin; Eri, eribulin; Tub, tubulin.

Fig. 4. Cevipabulin binds to the seventh site to induce tubulin degradation.

(A) HeLa cells were treated with 10 μM eribulin for 1 hour and then further treated with 1 μM cevipabulin for 16 hours. The α-tubulin protein level was detected by immunoblotting. Results are representative of three independent experiments. (B) Vectors expressing either wild-type (WT) or Y224G mutant GFP-tubulin were transfected to HeLa cells. After 24 hours, cells were treated with or without 1 μM cevipabulin for 16 hours. Then, the protein level of GFP-α-tubulin was detected by immunoblotting. Results are representative of three independent experiments. (C) Chemical structure of cevipabulin derivatives. (D) HeLa cells were treated with indicated compounds for 16 hours. Then, the protein level of α-tubulin was detected by immunoblotting. Results are representative of three independent experiments. (E) Indicated concentrations of compound 1 were incubated with tubulin (20 μM) for 10 min, and then the bound compound 1 was quantified by LC-MS/MS. This graph presented the molecular ratio of the compound 1:tubulin dimer. Data were shown as means ± SD of three independent experiments. (F) Vectors expressing either wild-type or Y224G mutant GFP-tubulin were transfected to HeLa cells. After 24 hours, cells were treated with or without 10 μM compound 1 for 16 hours. Then, the protein level of GFP-α-tubulin was detected by immunoblotting. Results are representative of three independent experiments. Cev, cevipabulin; 1, compound 1; 2, compound 2; Vin, vinblastine.

Fig. 5. Cevipabulin or compound 1 decreases tubulin stability to promote tubulin destabilization and degradation.

(A) The fluorescence of TPE-MI is enabled upon conjugation to a cysteine residue of a denatured protein. (B) Tubulin unfolding detected by TPE-MI. Tubulin was mixed with TPE-MI, and the indicated compounds for various times before fluorescence were detected (n = 3). (C) HeLa cells were treated with or without PYR-41 for 1 hour before treated with compound 1 for 16 hours. Protein level of α-tubulin was detected by immunoblotting. Results are representative of three independent experiments. (D) Close-up view of the seventh site in the cevipabulin-tubulin (black) and apo-tubulin (green, PDB code: 4i55) complex that were aligned on α2-tubulin. GTP, magenta sticks; cevipabulin, yellow sticks; side chain of α2-Y224, gray sticks. The collision of the α-T5 loop in the apo-tubulin complex to cevipabulin is marked with a red arrow. (E) Close-up view of the interaction between the nonexchangeable GTP and the α-T5 loop in the apo-tubulin (left) or the cevipabulin-tubulin (right) complex. Main chain of the α-T5 loop, sticks. Hydrogen bonds, red dashed lines. (F) Tubulin in buffer supplemented with 1 mM GDP was incubated with indicated compounds; both bound GTP and GDP were further quantified. Molecular ratio of the GTP:tubulin or GDP:tubulin dimer was shown. Data were presented as means ± SD of three independent experiments. *P < 0.05 (GTP) or #P < 0.05 (GDP), compound content as compared to the DMSO-treated group. Cev, cevipabulin; Vin, vinblastine; 1, compound 1; 2, compound 2. AU, arbitrary units.