Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d)

Abstract

HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte Ig-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including beta(2)-microglobulin (beta(2)m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the beta(2)m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the beta(2)m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-A resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCI recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G alpha3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/beta(2)m) and free forms of beta(2)m. Binding studies using beta(2)m-free MHCIs revealed differential beta(2)m-dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCIs, which may in turn regulate immune suppression.

Fig. 1.

Overall structure of the LILRB2 complex of HLA-G. (A) Ribbon diagram of the LILRB2/HLA-G1 complex. Red, LILRB2; cyan, HLA-G heavy chain; green, β₂m; blue, nonapeptide. LILRB2 uses two binding interfaces: D2-β₂m (site 1) and D1-α3 (site 2) domain. The stick models of the amino acids involved in the complex formation are shown as follows. Site 1: orange, D2 domain of LILRB2 (Trp-67, Asp-177, Asn-179, and Val-183); sky blue, β₂m (Lys-6 and Lys-91). Site 2: magenta, D1 domain of LILRB2 (Arg-36, Tyr-38, Lys-42, Ile-47, and Thr-48); blue, α3 domain (Phe-195, Tyr-197, and Glu-229). The descriptions of site 1 and site 2 apply to Figs. 1–4. (B) 2F_o − F_c map (green mesh) contoured at 1σ onto the stick model of the LILRB2/HLA-G complex (around Phe-195 and Tyr-197 of HLA-G).

Fig. 2.

Structural comparison of the LILRB2/HLA-G and LILRB1/HLA-A2 complexes. (A) The LILRB2/HLA-G complex is superimposed onto the LILRB1/HLA-A2 complex around the MHCI regions. The molecular surface of HLA-G is shown. Cyan, HLA-G heavy chain; green, β₂m; red, LILRB2; yellow, LILRB1. The D1 domain of LILRB2 has more binding interface on the α3 domain (site 2). The different interactions are observed in D2–β₂m interfaces (site 1). (B) This complex image is produced by rotating the image in A around the horizontal axis ≈90°. (C and E) The buried surface areas of LILRB2/HLA-G (C) and LILRB1/HLA-A2 (E). The buried surface areas were calculated by using the program SURFACE (CCP4 suite) with a 1.4-Å probe radius. Cyan, HLA heavy chain; green, β₂m; red, LILRB1 or LILRB2. The buried surfaces are shown in yellow. (D and F) NMR analysis of LILRB2–HLA-Cw7 and LILRB1–HLA-Cw7 interactions. The model orientation is similar to that of C and E. Mapping of amino acids whose ¹H-¹⁵N heteronuclear sequential quantum correlation peaks were perturbed upon complex formation is shown (orange, >0.09 ppm chemical shift changed; magenta, >50% intensity reduced; red, disappeared; green, <0.09 ppm chemical shift changed; white, unassigned). (D) Interaction of conformed (heavy chain/peptide/β₂m) (Left) and free (Right) forms of β₂m with LILRB2. (F) The same binding analysis as in D with LILRB1.

Fig. 3.

LILR binding interfaces (site 2) of the α3 domain of the LILRB2/HLA-G and LILRB1/HLA-A2 complexes. (A and C) LILRB2/HLA-G complex. (B and D) LILRB1/HLA-A2 complex. Cyan, HLA-G heavy chain; light blue, HLA-A2 heavy chain; green and light green, β₂m; magenta, LILRB2; yellow, LILRB1. (A and B) The binding interface around the 195–197 loop of HLA-G. (C and D) The binding interface around the cleft between the first 3₁₀ helix and the C strand of LILRBs.

Fig. 4.

SPR analyses. Binding of LILRB2 (Left) and LILRB1 (Right) to HLA-G heterotrimer (red lines) and β₂m-free HLA-G heavy chain (green lines). Heterotrimers and β₂m-free forms of MHCIs were immobilized on the sensor chip at ≈2,000 response units (RU). Black lines show the responses to the control protein (BSA).