Fig. 2: Cryo-EM structures of MCT8 and MCT10.

a Schematic representation of MCT transmembrane α-helices 1–6 (N-terminal domain, NTD) and 7–12 (C-terminal domain, CTD). The construct was engineered with an ALFA tag at the C-terminus to form a complex with its cognate nanobody NbALFA. Cryo-EM map of b ligand-free MCT8 in the outward-facing state (OFS), c T4-bound MCT8 in the outward-facing state (OFS), d T4-bound MCT10 in the inward-facing state (IFS). d, f Closed-up view on T4 binding site with the corresponding cryo-EM density for MCT8 and MCT10, respectively. g–i Zoomed views on the central gate region (corresponding to the white square in b, c, e), highlighting residues coordinating T4. j–l Cross sections of the surface of transporters (dotted line in b, c, e) indicating progressive closure of the gate, moving solvent accessibility of the substrate from the OUT to the IN compartment. To derive the position of residues in the long isoform of MCT8, 74 amino acids should be added (Supplementary Fig. 10). m T4 binding curves relative to MCT8 and its mutants measured by MST n T4 binding curves relative to MCT10 and its mutants measured by MST. Data are presented as mean ± SD (number of biological replicates, n = 4 in m with D424N, F336A, and in n with D396A; n = 3 for all the other samples). o Quantification of T4 uptake by HeLa cells overexpressing FLAG-MCT8 and its mutants (Supplementary Fig. 8). Data are presented as mean values ± SD (number of biological replicates, n = 7 with WT, n = 5 with N119A, and n = 3 for all the other samples). The dotted line indicates negligible endogenous MCT8 levels. Normalization was performed on overexpressed MCT8 + T4. P values are based on ordinary one-way ANOVA (**p = 0.0091, ***p = 0.0007, ****p < 0.0001). For the MCT8 mutants F115A p = 0.0091, R371A p < 0.0001, N119A p < 0.0001, F336A p = 0.0007, D424N p < 0.0001. Source data are provided as a Source Data File.