Fig. 3: Interactions between mCAT1and frRBD.
From: Structural insights into cationic amino acid transport and viral receptor engagement by CAT1
a Overview of the mCAT1/frRBD complex (mCAT1, forest green; frRBD, orange), showing contact regions on ECL3/ECL6 of mCAT1 and VRA region of frRBD. b Patch 1. Close-up of frRBD residues (Y94′, T129′, N133′, N137′, R131′) near mCAT1 ECL3 residues (K222, E221, E237). Dashed lines indicate potential hydrogen-bond/salt-bridge contacts. c Patch 2. Interdigitating contacts between frRBD residues (S114′, G116′, S118′, C117′, W136′, D120′) and mCAT1 residues (T231, N232, V233, K234, Y235, G236). Potential hydrogen bonds are indicated by dashed lines. d Patch 3. frRBD residues (R119′, E123′) near E60 (TM1) and K234 (ECL3) of mCAT1, and T126′ oriented toward R511 (ECL6). e Core interface. CH/π or π–π stacking of W136′ and P104′ from frRBD against Y235 and F224 of mCAT1. f Predicted changes in binding affinity (ΔΔG_affinity, kcal/mol) upon alanine substitution across three patches and the core. Positive values indicate reduced affinity. ΔΔGaffinity (kcal/mol)= ΔGwild-typle-ΔGmutant. g, Pseudovirus entry assay for selected frRBD alanine variants. The experiment was performed in three independent biological replicates with four parallel wells for each sample. Statistical comparisons versus WT were performed using two-sided t-tests (P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****; NS, not significant). Dashed lines indicate modeled hydrogen bonds or salt bridges. And NC corresponds to mutations in non-interfacial residues. Color code: mCAT1, forest green; frRBD, orange. RLU, Relative Luminescence Units; ECL, extracellular loop; ICL, intracellular loop. Statistics were performed using unpaired two-sided t-tests comparing each mutant to WT (n = 12 biological samples per group; 3 independent experiments, 4 independent samples per experiment). Exact P values: Y94A′, P = 0.7440; T129A′, P = 0.0138; R131A′, P = 0.2407; N133A′, P = 0.0986; N137A′, P = 0.9254; S144A′, P = 0.9024; S118A′, P = 0.4274; D120A′, P = 0.6974 R119A′, P = 0.0972; E123A′, P = 0.2092; T126A′, P = 0.2982; P104A′/W136A′, P = 0.0708.
