Benzimidazole resistance-associated mutations improve the in silico dimerization of hookworm tubulin: An additional resistance mechanism

Abstract

Background and aim: Mutations in the β-tubulin genes of helminths confer benzimidazole (BZ) resistance by reducing the drug's binding efficiency to the expressed protein. However, the effects of these resistance-associated mutations on tubulin dimer formation in soil-transmitted helminths remain unknown. Therefore, this study aimed to investigate the impact of these mutations on the in silico dimerization of hookworm α- and β-tubulins using open-source bioinformatics tools.

Materials and methods: Using AlphaFold 3, the α- and β-tubulin amino acid sequences of Ancylostoma ceylanicum were used to predict the structural fold of the hookworm tubulin heterodimer. The modeled complexes were subjected to several protein structure quality assurance checks. The binding free energies, overall binding affinity, dissociation constant, and interacting amino acids of the complex were determined. The dimer's structural flexibility and motion were simulated through molecular dynamics.

Results: BZ resistance-associated amino acid substitutions in the β-tubulin isotype 1 protein of hookworms altered tubulin dimerization. The E198K, E198V, and F200Y mutations conferred the strongest and most stable binding between the α and β subunits, surpassing that of the wild-type. In contrast, complexes with the Q134H and F200L mutations exhibited the opposite effect. Molecular dynamics simulations showed that wild-type and mutant tubulin dimers exhibited similar dynamic behavior, with slight deviations in those carrying the F200L and E198K mutations.

Conclusion: Resistance-associated mutations in hookworms impair BZ binding to β-tubulin and enhance tubulin dimer interactions, thereby increasing the parasite's ability to withstand treatment. Conversely, other mutations weaken these interactions, potentially compromising hookworm viability. These findings offer novel insights into helminth tubulin dimerization and provide a valuable foundation for developing anthelmintics targeting this crucial biological process.

Keywords: Ancylostoma; anthelmintic resistance; microtubules; soil-transmitted helminths.

Figure-1

Wild-type hookworm tubulin dimer folding prediction and structural quality assurance checks. AlphaFold3 was used to predict the 3D structure of the α-β-tubulin heterodimers (a). After protein refinement and energy minimization, the modeled dimers were assessed using VERIFY3D (b), PROCHECK’s Ramachandran Plot (c), and Protein Structure Analysis (d). Overall, hookworm tubulin folding predictions from the α and β subunit amino acid sequences were deemed satisfactory and accurate.

Figure-2

Pairwise structural alignment of the predicted folding of the hookworm tubulin α and β subunits with a previously described crystal structure of a mammalian tubulin complex (PDB ID No.: 1JFF). (a) α subunit and (b) β subunit. The analysis revealed that the modeled tubulin subunits have high structural similarity with their mammalian homologs, as determined using electron crystallography. The analysis was performed using the PDB Pairwise Structure Alignment Tool (https://www.rcsb.org/alignment).

Figure-3

The binding free energy (ΔG_bind) of the modeled hookworm tubulin heterodimers, both wild-type and mutant. The red dashed line indicates the ΔG_bind level of the wild-type dimer. Tubulin heterodimers with the E198K and E198V mutations in the β subunit significantly increased their ΔG_bind compared to the wild-type. Interactions between the α subunit and β subunits with these mutations are more thermodynamically favorable and stable. The ΔG_bind was estimated using HawkDock’s molecular mechanics/generalized born surface area platform (http://cadd.zju.edu.cn/hawkdock/).

Figure-4

Estimated binding site pocket volume of wild-type and mutated β-tubulin subunits. Most mutations reduced the benzimidazole binding site volume. The binding site volume was estimated using CAVER Web v. 1.2 server (https://loschmidt.chemi.muni.cz/caverweb/).

Figure-5

Root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent-accessible surface area (SASA), and radius of gyration plots of the wild-type and mutated tubulin dimers. (a) RMSD plot derived from 50,000 ps molecular dynamics simulations. (b) RMSF plot showing deviations per dimer residue. (c) SASA plot depicting the surface area of the tubulin dimer that is solvent accessible during the simulation period. (d) Radius of gyration plot showing the compactness of the dimer through the 50,000 ps simulation time.