Fig. 5: Identification of critical interaction residues between Dal81 and Stp2 responsible for pH alkalinization.
A AlphaFold 3 in silico docking simulations revealed that Dal81 and Stp2 formed two important hydrogen bonds (Dal81L405 - Stp2D526 and Dal81S406 - Stp2D528). Dal81 is visualized as yellowish brown cartoons, Stp2 as light blue. Predicted interaction structures are categorized by confidence intervals. B Schematics of C. albicans Dal81, with 21 AlphaFold3-predicted Stp2-interacting residues highlighted in red. C–E Immunoblots (C) of Dal81-Myc and Stp2-HA expression in indicated strains after 6 h in YNB + 1% CAA (initial pH 4.5). ImageJ quantification of Dal81-Myc (D) and Stp2-HA (E) band intensities. Data are mean ± SD (three biological replicates). Statistical significance via unpaired two-tailed Student’s t-test. * P < 0.05, ** P < 0.01; ns, not significant. F Alkalinization analysis of the specified strains was conducted following the procedures described in Fig. 1B. G Stp2-HA failed to co-immunoprecipitate with Dal81m21-Myc. Strains co-expressing Stp2-HA and Dal81m21-Myc were grown in YNB + 1% CAA (initial pH4.5). Whole cell extracts were prepared under nondenaturing conditions, and immunoprecipitated with anti-Myc beads. Precipitates were immunoblotted with anti-HA or anti-Myc antibodies. R1 and R2 denote biological replicates. H Growth curves of indicated strains in medium 199 (initial pH 4.0) at 37 °C, with OD600 measured every 15 min (BioTek Synergy 2 Multi-mode Microplate Reader). Data are mean ± SD (three independent biological replicates). Statistical analysis via one-way ANOVA with Tukey’s multiple comparison test at 24 h. * P < 0.05, ** P < 0.01. I Alkalinization assay (as in Fig. 1B) showing pH modulation defects in DAL81m21/dal81Δ. Source data for (C–E, G, H) are provided in the Source Data file.
