Extended Data Fig. 6: RPL20B central domain binds to PD-L1 IgV domain.
From: Profiling phagosome proteins identifies PD-L1 as a fungal-binding receptor
a, Protein sequence alignment of RPL20B homologs from different fungal species. Sequence of the central domain is highlighted in the shaded box. b, Three-dimensional structure of RPL20B-PD-L1 complex by molecular modeling. The interaction between the two molecules is shown in a surface representation. The putative contacting residues from RPL20B are shown in red color. c, ELISA assay assessing the binding of RPL20B-CDP to B7 family Fc fusion proteins with technical triplicates. d, Microscale thermophoresis assay determining the binding affinity of hPD-L1-Fc to N-terminal biotin-modified RPL20B-CDP complexed to avidin. n = 3 biological replicates. e, Binding of hPD-L1-Fc to streptavidin beads loaded with biotin-RPL20B-CDP peptide were assessed by flow cytometry. f, g, Recombinant human PD-L1 Fc fusion proteins (f) or native mouse PD-L1 in GM-CSF-primed BMDM lysate (g) were pulled down with RPL20B-CDP-loaded beads and the immunoblots were probed with anti-human or mouse PD-L1 antibodies. h, ELISA assay determining binding of Fc fusion proteins of the two individual immunoglobulin-like extracellular domains of PD-L1 to RPL20B-CDP with technical triplicates. i, Flow cytometry assessing the binding of hPD-L1-Fc to yeast expressing C. albicans RPL20B with the indicated mutations. n = 3 biological replicates. j, A closeup of the interacting amino acid residues from C. albicans RPL20B (red) to those from human PD-L1 (green) is shown in a stick model. The proteins, RPL20B and PD-L1 are shown in ribbon representation. MFI, mean fluorescent intensity. ΔMFI, MFI minus that of the respective Fc control. Data are mean + S.E.M. (c, e, h, i) or mean ± S.D. (d). Data are representative of 3 independent experiments (c, e, f, g, h, i). P values by one-way ANOVA with Tukey’s multiple comparisons tests (c, e, i). ****P < 0.0001.
