The structure and stability of the monomorphic HLA-G are influenced by the nature of the bound peptide

Abstract

The highly polymorphic major histocompatibility complex class Ia (MHC-Ia) molecules present a broad array of peptides to the clonotypically diverse alphabeta T-cell receptors. In contrast, MHC-Ib molecules exhibit limited polymorphism and bind a more restricted peptide repertoire, in keeping with their major role in innate immunity. Nevertheless, some MHC-Ib molecules do play a role in adaptive immunity. While human leukocyte antigen E (HLA-E), the MHC-Ib molecule, binds a very restricted repertoire of peptides, the peptide binding preferences of HLA-G, the class Ib molecule, are less stringent, although the basis by which HLA-G can bind various peptides is unclear. To investigate how HLA-G can accommodate different peptides, we compared the structure of HLA-G bound to three naturally abundant self-peptides (RIIPRHLQL, KGPPAALTL and KLPQAFYIL) and their thermal stabilities. The conformation of HLA-G(KGPPAALTL) was very similar to that of the HLA-G(RIIPRHLQL) structure. However, the structure of HLA-G(KLPQAFYIL) not only differed in the conformation of the bound peptide but also caused a small shift in the alpha2 helix of HLA-G. Furthermore, the relative stability of HLA-G was observed to be dependent on the nature of the bound peptide. These peptide-dependent effects on the substructure of the monomorphic HLA-G are likely to impact on its recognition by receptors of both innate and adaptive immune systems.

Fig. 1

Structure of pHLA-G complexes. (a and b) Side views of the HLA-G^KGPPAALTL complex (a) and the HLA-G^KLPAQFYIL complex (b) showing 2.4- and 1.7-Å omit maps, respectively (contoured at 1σ). The α2 helix has been removed for clarity.

Fig. 2

Conserved interactions between peptide and HLA-G. The polar and non-polar contacts of the P1 residue (a, c, e) and the P9 residue (b, d, f) of each pHLA-G complex are shown. (a and b) HLA-G^KGPPAALTL. (c and d) HLA-G^KLPAQFYIL. (e and f) HLA-G^RIIPRHLQL. H-bonds and salt bridges are represented by dashed lines, and water molecules are represented by red spheres.

Fig. 3

Peptide conformation. (a) Comparison of the conformations of the KGPPAALTL peptide (green) and the RIIPRHLQL peptide (purple) when presented by HLA-G highlighting the similarity between the two peptides. (b) Comparison of the conformations of the KLPAQFYIL peptide (orange) and the RIIPRHLQL peptide (purple) when presented by HLA-G highlighting the central bulge in KLPAQFYIL.

Fig. 4

Orientation of His70. The orientation of His70 side chain is altered by the presence of a His at P6 of RIIPRHLQL (c) compared with that seen in the presence of KGPPAALTL (a) and KLPAQFYIL (b). An overlay of the three peptides is shown in (d).

Fig. 5

Comparison of the three pHLA-G complexes. Top view (a) and side view (b) of HLA-G showing the backbones of RIIPRHLQL (purple), KGPPAALTL (green) and KLPAQFYIL (orange). The α2 helix has been removed from (b) for clarity. (c) A 0.9-Å shift in the α2 helix centered at V152 is seen in the HLA-G^KLPAQFYIL complex (orange) when compared with HLA-G^RIIPRHLQL (purple) and HLA-G^KGPPAALTL (green).

Fig. 6

Conserved peptide contacts. Polar contacts between the KGPPAALTL peptide and HLA-G are represented by dashed lines shown in yellow for conserved contacts and in black for non-conserved contacts. Water molecules are represented by red spheres.

Fig. 7

Thermal stability of pHLA-G complexes. The ellipticity at 218 nm was measured as temperature increased from 20 to 90 °C. The data were normalised to determine the midpoint of thermal denaturation (T_m). Samples were diluted to 5 and 10 μM in 10 mM Tris, pH 8.0, containing 150 mM NaCl. The complexes were measured twice at both 5 and 10 μM, and the T_m was averaged over the four experiments. The figure is representative of denaturation at 5 μM. Data for HLA-G^RLPKDFRIL, HLA-G^KLPAQFYIL, HLA-G^KGPPAALTL and HLA-G^RIIPRHLQL are shown in red, orange, green and purple, respectively.