Metal-triggered conformational reorientation of a self-peptide bound to a disease-associated HLA-B*27 subtype

Abstract

Conformational changes of major histocompatibility complex (MHC) antigens have the potential to be recognized by T cells and may arise from polymorphic variation of the MHC molecule, the binding of modifying ligands, or both. Here, we investigated whether metal ions could affect allele-dependent structural variation of the two minimally distinct human leukocyte antigen (HLA)-B*27:05 and HLA-B*27:09 subtypes, which exhibit differential association with the rheumatic disease ankylosing spondylitis (AS). We employed NMR spectroscopy and X-ray crystallography coupled with ensemble refinement to study the AS-associated HLA-B*27:05 subtype and the AS-nonassociated HLA-B* 27:09 in complex with the self-peptide pVIPR (RRKWRRWHL). Both techniques revealed that pVIPR exhibits a higher degree of flexibility when complexed with HLA-B*27:05 than with HLA-B*27:09. Furthermore, we found that the binding of the metal ion Cu²⁺ or Ni²⁺, but not Mn²⁺, Zn²⁺, or Hg²⁺, affects the structure of a pVIPR-bound HLA-B*27 molecule in a subtype-dependent manner. In HLA-B*27:05, the metals triggered conformational reorientations of pVIPR, but no such structural changes were observed in the HLA-B*27:09 subtype, with or without bound metal ion. These observations provide the first demonstration that not only major histocompatibility complex class II, but also class I, molecules can undergo metal ion-induced conformational alterations. Our findings suggest that metals may have a role in triggering rheumatic diseases such as AS and also have implications for the molecular basis of metal-induced hypersensitivities and allergies.

Keywords: HLA-B*27; NMR spectroscopy; X-ray crystallography; ankylosing spondylitis; autoimmune disease; autoimmunity; conformational change; crystal structure; ensemble refinement; hypersensitivity; major histocompatibility complex (MHC); nuclear magnetic resonance (NMR); peptide dynamics.

Figure 1.

Peptide binding to HLA-B*27 subtypes. According to previously published work (9), the pVIPR peptide binds to the B*27:09 subtype in the CC conformation shown in orange. The view is through the α2-helix onto the α1-helix. The peptide is drawn as a ribbon and arginine residues in stick representation (A), whereas a dual binding mode (CC/NC) was observed for B*27:05. The CC conformation is shown in magenta, and the NC conformation is in cyan (B), where pArg-5 forms a salt bridge (red dashed lines) to HC residue Asp-116 in the NC-binding mode. This contact is precluded in B*27:09 (His-116) due to electrostatic repulsion. SOFAST-¹H,¹⁵N-HMQC spectra of both pVIPR-HLA-B*27 complexes containing ¹⁵N,¹³C-labeled Arg at positions 1, 2, 5, and 6 of the peptide are shown in B*27:09 (C) and B*27:05 (D); the appearance of natural abundance signals from the HC and β₂m in the center of the spectra is due to the long experiment duration. Despite the broad lines, the spectrum for B*27:09-complexed pVIPR appears as expected, with three backbone peaks for pArg-2, pArg-5, and pArg-6 (black) and four peaks for the side chains of the four Arg residues mentioned above (red). In the spectrum of pVIPR-B*27:05, however, only two peaks for backbone resonances are visible (black) instead of the expected five; in addition, only four peaks for the side chains of Arg residues (red) can be observed (six are expected).

Figure 2.

pVIPR conformations and metal binding sites. HLA-B*27 complexes are shown in gray cartoon and the respective peptide in stick representation; the polymorphic residue at position 116 at the floor of the peptide binding groove is drawn in green, and the view is through the α2-helix onto the α1-helix. A, in the 100 K as well as in the RT structure, B*27:05 displays the peptide (pink) exclusively in the NC conformation. The NC binding mode allows the establishment of a salt bridge (red dashed lines) between pArg-5 and the HC residue Asp-116. B, a dual peptide-binding mode (CC/NC; same color coding as in Fig. 1A) is induced by Cu²⁺ or Ni²⁺ ions bound to B*27:05. The Cu²⁺ cations are depicted as yellow spheres. C, in contrast, B*27:09 displays pVIPR in the CC-binding mode, irrespective of the presence or absence of metal ions. The HC residue His-116 precludes a direct contact with pArg-5 of the peptide.

Figure 3.

Coordination of Cu²⁺ and Ni²⁺ bound to pVIPR-HLA-B*27 complexes. A given cation is coordinated identically when bound to pVIPR complexes of the two subtypes, but there are differences in coordination between the two metal ions. Anomalous difference maps are contoured at 8 σ in all panels around the respective metal ion. A, view along the α1-helix toward the C terminus of the peptide. Only the two C-terminal peptide residues (pHis-8, pLeu-9) are shown (violet). The coordination of Cu²⁺ in B*27:05 depicts the involvement of pHis-8. Further coordinating residues are Glu-76 (side chain conformation B), two water molecules shown as red spheres, and His-197 from a neighboring HLA-B*27 molecule. B, the coordination of Ni²⁺ in B*27:05 is shown. Instead of Glu-76, an additional water molecule contributes to coordinate the cation.

Figure 4.

Results of the ensemble refinement performed for the structures of pVIPR bound to B*27:05 and B*27:09, respectively. For clarity, the HC and β₂m are not shown. The peptide and the polymorphic residue at position 116 are displayed in a stick representation. The peptide is color-coded by decreasing temperature factor from red to blue. A, pVIPR-B*27:05 shows increased dynamics; B, pVIPR shows a lower degree of flexibility when presented by B*27:09. C, view of A rotated by 90°. pVIPR-B*27:05 is oriented such that an approaching TCR would “see” the peptide. D, view of B rotated by 90°. pVIPR-B*27:09 is oriented such that an approaching TCR would “see” the peptide.

Figure 5.

Results of the ensemble refinement performed for the structures of pVIPR bound to B*27:05 in the presence of Cu²⁺. For clarity, the HC and β₂m are not shown. The peptide and the polymorphic residue at position 116 are displayed in a stick representation. The peptide is color-coded by decreasing temperature factor from red to blue. A, pVIPR-B*27:05-Cu²⁺ in NC conformation; B, pVIPR-B*27:05-Cu²⁺ in the CC conformation. C, view of A rotated by 90°, such that an approaching TCR would “see” the peptide. D, view of B rotated by 90°, such that an approaching TCR would “see” the peptide.

Figure 6.

Differential presentation of pVIPR and closely related ligands by the B*27:05 subtype. All B*27:05-bound ligands are shown in identical orientation, through the α2-helix (removed) onto the α1-helix (gray). A, pVIPR bound in the NC conformation. Note the establishment of a salt bridge between pArg-5 and the HC residue Asp-116, indicated by red dashed lines. B, pVIPR bound in a dual conformation (NC in cyan, CC in magenta) triggered by a Cu²⁺ ion in the vicinity of pHis-8. C, pLMP2 bound in the NC conformation. Note the molecular mimicry between this structure and that of pVIPR shown in Fig. 3A. D, pVIPR with pArg-5 replaced by citrulline. This neo-antigen is bound in the CC conformation, closely resembling that found for pVIPR-CC (B), because a contact between the side chain of citrulline and Asp-116 cannot be established.