Fig. 2: Structural analysis of LAG3-MHC-II binding interfaces and stoichiometry.

a LAG3-MHC-II binding interfaces on the structure of scLAG3-MHC-II are indicated with black boxes. The Loop 1 insertion in LAG3 D1 was disordered in the structure and is indicated by a dotted line, and the residues of Loop 2 are shown as blue spheres. The MHC-II α and β chains are colored salmon and violet, respectively, and mLAG3 is colored cyan. The left zoom panel depicts Loop 2 interactions with the α2 and β2 subdomains of I-A^b. LAG3 residues associated with loss-of-binding mutations are labeled in red, with asterisks designating mutations that were tested in this study. The Cα positions of G103R and P111A mutations are marked with red spheres. The right zoom panel depicts the charge-complementary surface between strand-connecting loops of LAG3 and the α2 domain of I-A^b. b SPR isotherms comparing the binding of mLAG3-Fc and mLAG3^P111A-Fc to immobilized I-A^b. The mLAG3 and mLAG3^P111A proteins were each produced as N-terminal fusions to human IgG1 Fc. c SPR isotherms comparing the binding of mLAG3 and mLAG3^G103R or mLAG3^R125A to immobilized I-A^b, and the binding of mLAG3 to I-A^b containing E198A and E199A mutations. d Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) was used to measure the solution molecular weight of scLAG3-MHC-II, which corresponds to a 2:2 complex. The molecular weight indicated is an average of three independent experiments. e A 2:2 complex of mLAG3^D12 (cyan and teal) bound to I-A^b (violet/salmon), generated through crystallographic symmetry, is depicted as it may occur between two cells. Engagement of MHC-II by LAG3 dimers separates the MHC-II molecules by ∼105 Å. APC: antigen-presenting cell. f A 2:2 model of LAG3-MHC-II was generated by superimposing two copies of scLAG3-MHC-II onto the structure of dimeric mouse LAG3 (PDB 8DGG)¹⁶.LAG3 is in green and MHC-II is in violet/salmon. Source data are provided as a Source Data file.