The molecular basis for peptide repertoire selection in the human leucocyte antigen (HLA) C*06:02 molecule

Abstract

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Keywords: X-ray crystallography; autoimmunity; major histocompatibility complex (MHC); mass spectrometry (MS); psoriasis.

Figure 1.

Analysis of peptides eluted from HLA-C*06:02. A, length distribution of HLA-C*06:02 presented peptides. B, peptide preference motif obtained for peptide nonamers for membrane bound HLA-C*06:02. Dominant, strong, and preferred amino acids occur at frequencies of >30, >20, and >10%, respectively. C, sequence Logo of nonomer peptides of membrane-bound HLA-C*06:02. Enriched amino acids are shown above and depleted are shown below. Height of amino acids are proportional to frequency of occurrence. Sequence Logos were generated with Seq2Logo server as a p-weighted Kullback-Leibler logo. D, overlap of peptide nonamers from membrane-bound and soluble HLA-C*06:02. E, peptide preference motif obtained for peptide nonamers from soluble HLA-C*06:02. F, sequence Logo of nonomer peptides of soluble HLA-C*06:02.

Figure 2.

Overall structures of the peptide-binding grooves of HLA-C*06:02-ARTE, ARFN, and ADAMTSL5. The α2 helix has been removed for clarity. In gray is the simulated annealing F_o − F_c omit electron density surrounding the peptide ligands, contoured at 3σ. A, the HLA-C*06:02 peptide-binding groove is represented as a schematic (teal) with the ARTE peptide as sticks (yellow). B and C, the ARFN peptide was observed in two conformations in the asymmetric unit. B, the ARFN-1 confirmation with the HLA represented as a schematic (blue) and the peptide as sticks (green). C, the ARFN-2 conformation with the HLA represented as a schematic (lime green) and the peptide as sticks (blue). D, the ADAMTSL5 peptide. The HLA is represented as a schematic (green) and the peptide as sticks (wheat). E, overlay of Cα traces of HLA-C*06:02 structures. The regions of difference at the β1-β2 loop and α2 helical hinge are labeled. F, overlay of the HLA-C*06:02 peptide ligands.

Figure 3.

Structure of HLA-C*06:02-ARTE. A, the ARTE peptide within the HLA-C*06:02 peptide-binding groove. Residues 1–126 of the HLA are represented as a schematic (teal) and the ARTE peptide is shown as yellow sticks. The pockets of the peptide-binding groove are represented as gray discs and labeled A–F. B, the B-pocket of HLA-C*06:02. Contacts between the P2 arginine anchor (yellow sticks) and side chains of HLA-C*06:02 residues (orange sticks). C, the E-pocket of HLA-C*06:02. Contacts between the P7 arginine anchor (yellow sticks) and side chains of HLA-C*06:02 residues (orange sticks). D, the F-pocket of HLA-C*06:02. Contacts between the PΩ leucine anchor (yellow sticks) and side chains of HLA-C*06:02 (orange sticks).

Figure 4.

Comparison of the two conformations of the ARFN peptide observed in the crystal structure. Shown is the first conformation of ARFN (ARFN-1) represented as sticks (green) with the HLA represented as a schematic (gray) with HLA side chains contacting the peptide shown as sticks (pink). The second conformation of the ARFN (ARFN-2) peptide is represented as sticks (blue) the HLA represented as a schematic (gray) with HLA side chains contacting the peptide shown as sticks (pink). A, conservation of contacts between the HLA and the ARFN-1 and ARFN-2 peptides at the P2-Arg position. B, conservation of contacts between the HLA and the ARFN-1 and ARFN-2 peptides at the PΩ-Val position. C, the ARFN-1 and ARFN-2 conformations differ in the orientation of their P7-Arg side chain. The P7-Arg of ARFN-1 forms a salt-bridge with Asp⁹, whereas the P7-Arg of ARFN-2 is shifted 5.8 Å and forms a salt-bridge with Asp¹¹⁴. D, plasticity of Trp⁹⁷ was observed between the HLA-C*06:02-ARTE and -ARFN structures. The ARTE, ARFN-1, and ARFN-2 peptides are shown as yellow, green, and blue sticks, respectively. The Trp⁹⁷ observed in the ARTE complex (orange sticks) is rotated 140° compared with the Trp⁹⁷ of the ARFN complex (pink sticks).

Figure 5.

Structure of HLA-C*06:02-ADAM peptide analogue (VRSRR-abu-LRL). A, view of the ADAMTSL5 peptide analogue (VRSRR-abu-LRL) within the HLA-C*06:02 peptide-binding groove, with the α2 helix removed for clarity. HLA-C*06:02 residues 1–126 are represented as a schematic (green) with side chains within 3.4 Å represented as sticks (blue). The ADAMTSL5 peptide is represented sticks (wheat). B, overlay of P7-Arg of HLA-C*06:02-ARTE, represented as sticks (gray), with the P7-Leu of ADAMTSL5 represented as sticks (wheat). Plasticity of Trp⁹⁷ was observed between the HLA-C*06:02-ARTE and -ADAMTSL5 structures. The ARTE peptide is colored gray, the ADAMSTL5 peptide is colored wheat. The Trp⁹⁷ observed in the ARTE complex (gray sticks) is rotated 114° compared with the Trp⁹⁷ of the ADAMSTL5 complex (blue sticks).

Figure 6.

Comparison of available HLA-C structures. A, Cα trace overlay of HLA-C*06:02 (teal), HLA-C*03:04 (PDB 1EFX) (green), HLA-C*04:01 (PDB 1QQD) (pink), HLA-C*05:01 (PDB 5VGD) (purple), HLA-C*07:02 (PDB 5VGE) (yellow), HLA-C*08:01 (PDB 4NT6) (wheat) and HLA-B*27:05 (PDB 3BP4) (blue). B–H, surface electrostatics of: HLA-C*06:02 ARTE (B), HLA-C*04:01 (C), HLA-C*03:04 (D), HLA-C*05:01 (E), HLA-C*08:01 (F), HLA-C*07:02 (G), and HLA-B*27:05 (H). B- and E-pockets are shown as yellow circles. Structures were generated using APBS Tools plug-in within PyMOL.

Figure 7.

A comparison of the anchor pockets of HLA-C*06:02 with other HLA-C alleles. A, sequence alignment of the B-, E-, and F-pockets of common HLA-C molecules. Residues conserved with HLA-C*06:02 are shown as a dash (–). B–G, a structural comparison of the anchor pockets of HLA-C*06:02 and HLA-C*04:01 (PDB 1QQD). The HLA-C*06:02-ARTE peptide is represented as yellow sticks. The alternate conformation of the P7-Arg of the ARFN peptide is shown as blue sticks. The HLA-C*06:02 pocket residues are shown as teal sticks and van de Waals surface (VDW). The HLA-C*04:01 peptide is represented as pink sticks. The HLA-C*04:01 pocket residues are shown as purple sticks and VDW surface. Panels B and C, the B-pocket. Panels D and E, the E-pocket. Panels F and G, the F-pocket.