Fig. 3

MHCI can bind directly to PDZ domains in vitro. a Schematic showing the exons that encode the classical MHCI H2-K^b. A signal sequence (S) is followed by exons encoding the three extracellular alpha domains, a single exon encoding the transmembrane domain, and three exons (6, 7, and 8) encoding the intracellular domain (not drawn to scale). Bottom, amino acid sequence of exons 6, 7, and 8 in H2-K^b. Putative PDZ ligand motifs are underlined. Exon 6 encodes the overlapping ligand motifs GDYA, DYAL, and YALA, exon 7 encodes the overlapping ligand motifs TSDL and DLSL, and exon 8 encodes the C-terminal motif HSLA. Only the overlapping motifs DCKV and KVMV span an exon/intron boundary. Notably, KVMV is also among the least conserved putative PDZ ligand motifs in H2-K^b, since it is not detected in this position in any other mouse MHCI. Intronic structure, NCBI. Adapted from [15]. b Schematic of in vitro binding assay. Different recombinant PDZ domain peptides are bound to each membrane spot, and the membrane is panned with GST-tagged recombinant cytoplasmic domains from specific MHCI proteins. A direct interaction between the cytoplasmic domain of MHCI and a given PDZ domain will be apparent as a black spot after visualization of bound anti-GST antibodies. c The cytoplasmic domain of the classical MHCI H2-K^b can bind directly to PDZ1 and to a lesser extent PDZ4+5 of MAGI-1, but not to PDZs 2 or 3 of the same protein. See Additional file 4: Figure S4B&D for validation of the identity of H2-K peptides, and S4E-F for titration of H2-K^b binding. d The cytoplasmic domain of the classical MHCI H2-K^b does not bind to PDZ1+2 (expressed together) or PDZ 3 of SAP97. e MAGI-1 binding by recombinant cytoplasmic domains derived from different classical and nonclassical MHCI proteins. Notably, the ability to bind to PDZ1 of MAGI-1 correlates with the presence of a class 1 PDZ ligand motif, which is present in H2-K^b and H2-T22, but not H2-D or H2-T23 (see f). f Amino acid sequences of the cytoplasmic domains of mouse and human MHCIs, aligned in ClustalW. Highlighted, putative class 1 PDZ ligand motifs (consensus [X – S/T – X – V/L]) that match MAGI-1 PDZ1’s binding preferences [80–82]. g Competition assays show that preincubation of column-bound MAGI-1 PDZ1 with cytoplasmic peptides derived from H2-T22, which can bind, but not H2-D, which cannot, precludes subsequent binding by peptides derived from H2-K. Left, pure H2-K not applied to a column; “none”, preincubation in buffer alone. H2-K was detected using an antibody against the cytoplasmic domain of H2-K (see Methods). Bottom, anti-His antibody shows eluted MAGI-1 peptide (arrow). h A point mutation in the lone class 1 PDZ ligand motif, TSDL, in the cytoplasmic domain of H2-K^b attenuates binding to MAGI-1 PDZ1. The location of the mutated residue (T329) is shown in (a)