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[Preprint]. 2023 Dec 4:2023.12.01.569627.
doi: 10.1101/2023.12.01.569627.

Experimental structures of antibody/MHC-I complexes reveal details of epitopes overlooked by computational prediction

Lisa F Boyd  1 , Jiansheng Jiang  1 , Javeed Ahmad  1 , Kannan Natarajan  1 , David H Margulies  1
Affiliations

Experimental structures of antibody/MHC-I complexes reveal details of epitopes overlooked by computational prediction

Lisa F Boyd et al. bioRxiv. .

Update in

Abstract

Monoclonal antibodies (mAb) to major histocompatibility complex class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I antigen-binding fragments (Fab) bound to peptide/MHC-I/β2m (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAb, and an anti-β2-microglobulin (β2m) mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer (AF-M) showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

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Conflict of interest statement

Authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.
X-ray structure of complex between Fab34-5-8 and H2-Dd reveals footprint on a2 domain. Illustration in cartoon and partially transparent surface representation of (A) complex of Fab34-5-8 with H2-Dd. H2-Dd a (Heavy) chain, medium slate blue, β2m (light coral), peptide (yellow), Fab H chain, dark cyan, Fab L chain, red; (PDB 8TQ8); (B) pMHC complex alone; (C) Fab34-5-8 H/L alone; (D) H2-Dd α1α2 and peptide rotated to allow visualization of interface; (E) Fab34-5-8 combining site rotated to permit visualization of H (cyan) and L (red) contact residues. (F) structure guided alignment of the α2 domains of the indicated mouse MHC-I molecules. Secondary structure elements above the alignment determined from PDB 3ECB. Contacts of Fab H and L chains are indicated (downward cyan (H) and magenta (L) arrows and brown (both) ovals), and residues that both contact the Fab and are polymorphic are indicated by upward blue arrows. Alignment was performed with Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/ (91) and illustration prepared with ESPript 3.0 https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi (92).
Fig. 2.
Fig. 2.
X-ray structure of complex between Fab34-2-12 and H2-Dd reveals footprint on α3 domain. Illustration in cartoon and partially transparent surface representation of (A) complex of Fab34-2-12 with H2-Dd (Colors of indicated chains as in Fig. 1); (B) pMHC (H2-Dd) complex alone; (C) Fab34-2-12 H/L alone; (D) H2-Dd α3 alone with VHVL rotated to visualize contacts; (E) pMHC rotated to allow visualization of Fab contacts; (F) Fab rotated to allow visualization of contacts to pMHC; (G) alignment of amino acid sequences of α3 domains of the indicated murine MHC-I molecules, performed as described in legend to Fig. 1. Contacts of Fab34-2-12 H and L chains are indicated, as are contacts to CD8α and CD8β as determined in PDB 3DMM (48).
Fig. 3.
Fig. 3.
X-ray structure of complex between Fab28-14-8 and H2-Db reveals footprint on α2 and α3 domains. Illustration in cartoon and partially transparent surface representation of (A) Fab28-14-8 with H2-Db (Colors of indicated chains as in Fig. 1); (B) pMHC complex alone; (C) Fab28-14-8 H/L alone; (D) H2-Dd rotated to visualize contacts to Fab; (E) Fab rotated to allow visualization of contacts to pMHC; (F) alignment of amino acid sequences of α2 and α3 domains of the indicated murine MHC-I molecules, performed as described in legend to Fig. 1. Contacts of Fab28-14-8 H and L chains are indicated, as are contacts of H2-Dd to CD8α and CD8β as determined in PDB 3DMM (48).
Fig. 4.
Fig. 4.
X-ray structure of complex between FabS19.8 and H2-Dd/β2mb reveals footprints on α1 and α2 domains as well as on β2m. Illustration in cartoon and partially transparent surface representation of (A) complex of FabS19.8 and H2-Dd/β2mb (Colors of indicated chains as in Fig. 1); (B) pMHC complex alone; (C) FabS19.8 H/L alone; (D) H2-Dd/β2mb rotated to visualize contacts to Fab; (E) Fab rotated to allow visualization of contacts to pMHC; (F) alignment of amino acid sequences of α1 and α2 domains of the indicated murine MHC-I molecules; (G) alignment of the indicated β2m sequences; (H) close-up illustration of sticks representation of β2mb addressing surface of S19.8 H chain residues 102–104; (I) model of clash encountered by substitution of β2m Ala85 to Asp, superposed on structures illustrated in (H).
Fig. 5.
Fig. 5.
Comparison of X-ray structures of Fab/MHC complexes to AF-M models reveals differences in experimental vs. computed docking sites of the Fab. AF-M predictions of Fab/MHC complexes were accomplished as described in Materials and Methods, and the X-ray determined models were compared to the “best_model” from each of the AF-M predictions. The indicated MHC-I heavy chains of the structures were superposed and the complete models were displayed in ChimeraX. Panels (A, D, G, J) show the X-ray structures and panels C, F, I, and J the AF-M models, while panels B, E, H, and K show the superpositions. MHC H chain is purple and L chain coral, X-ray Fab H chain is cyan and L chain red. For the AF-M models MHC colors are the same, but Fab H chain is dark grey and L chain is light grey. For the superposed Fab/MHC complex structures and predictions, we calculated the difference in the location of the center of mass (COM) of each Fab H and L chain. For 34-5-8 (B), H chain differed by 86.2 Å and L chain by 100.0 Å). Similarly, for 34-2-12 (E), H and L differed by 95.1 and 112.9 Å, respectively; for 28-14-8 (H), H and L by 81.2 and 88.4 Å; for S19.8 (K), by 81.2 and 88.4 Å. MHC-I, β2m, and peptide COM in all complexes differed by less than 1.6 Å.
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