Structural aspects of chemical modifications in the MHC-restricted immunopeptidome; Implications for immune recognition

Abstract

Significant advances in mass-spectroscopy (MS) have made it possible to investigate the cellular immunopeptidome, a large collection of MHC-associated epitopes presented on the surface of healthy, stressed and infected cells. These approaches have hitherto allowed the unambiguous identification of large cohorts of epitope sequences that are restricted to specific MHC class I and II molecules, enhancing our understanding of the quantities, qualities and origins of these peptide populations. Most importantly these analyses provide essential information about the immunopeptidome in responses to pathogens, autoimmunity and cancer, and will hopefully allow for future tailored individual therapies. Protein post-translational modifications (PTM) play a key role in cellular functions, and are essential for both maintaining cellular homeostasis and increasing the diversity of the proteome. A significant proportion of proteins is post-translationally modified, and thus a deeper understanding of the importance of PTM epitopes in immunopeptidomes is essential for a thorough and stringent understanding of these peptide populations. The aim of the present review is to provide a structural insight into the impact of PTM peptides on stability of MHC/peptide complexes, and how these may alter/modulate immune responses.

Keywords: T cell receptor (TCR); epitope; human leucocyte antigens (HLA); immune responses; immunopeptidome; major histocompatibility complex (MHC); post-translation modification.

FIGURE 1

The overall structures of MHC-I and MHC-II complexes are similar. (A) Despite the similarity of their overall three dimensional folds, there are some significant differences between MHC-I and MHC-II molecules. Left panel: The polymorphic MHC-I heavy chain, colored in grey, comprises the peptide binding domain (PBD or α1-α2 domain) and an additional Ig-like domain denominated α3. Furthermore, the almost invariable Ig-like β₂-microglobulin (β2m) domain, colored in green, binds non-covalently between the α1-α2 and α3 entities, stabilizing the overall structure of MHC-I/peptide complexes (pMHC). The peptide is colored in yellow. Right panel: MHC-II molecules consist of the two polymorphic α and β chains (in grey and green, respectively), each one comprising half of the PBD and each one with an additional Ig domain that separates the PBD from the cell membrane. The peptide is colored in yellow. (B) T cell receptors bind on the top of pMHC molecules, interacting with both the presented peptides and the MHC chains. The surfaces of both TCR and pMHC are semitransparent. Left panel: The α and β chains of the MHC-I-restricted TCR are colored light blue and orange, respectively, while the surfaces of the heavy chain and β₂m are in light violet and light green, respectively. The side chains of the presented peptide are represented by yellow spheres. Nitrogen and oxygen atoms are in blue and red, respectively. Classical examples of canonical peptide anchor residues are indicated by arrows, with their side chains buried within specific MHC pockets. The N- and C-termini of MHC-I peptide-binding clefts are closed, restricting most often the length of the bound peptides to 8–10 amino acids. Right panel: The α and β chains of the MHC-II-restricted TCR are in light blue and orange, respectively. The three dimensional structures of the ternary complexes derveal that MHC-II-restricted TCRs most often bind very similarly to their cognate pMHC-II complexes compared to MHC-I-restricted TCRs. In contrast to a majority of MHC-I-restricted peptides, the flanking residues of most MHC-II-restricted epitopes are protruding our from both the N- and C-termini of the MHC-II cleft and are more solvent accessible. All figures were created using PyMOL (The PyMOL Molecular Graphics System, Version 2.3.0, Schrödinger, LLC). All figures were created using the TCR/pMHC ternary structures determined in 2BNR.pdb and 1ZGL.pdb.

FIGURE 2

Glycosylation occurs most often in the middle section of the peptide creating a neoantigen that can break tolerance and select for different T cell repertoires (A). Comparative analyses of the crystal structures of H-2D^b in complex with the immunodominant Sendai virus epitope FAPGNYPAL (1CE6. pdb) colored in yellow and with the glycopeptide analog K2G with a glycan at p4 (1QLF.pdb), colored in green) (Glithero et al., 1999) reveal the similarity of the peptide backbones. In contrast, the protruding glycan moiety (in blue) may impair/modulate TCR recognition (B). Comparison of the crystal structures of H-2K^b in complex with the vesicular stomatitis virus (epitope RGYVYQGL (2VAA.pdb, in yellow) (Fremont et al., 1992) and the glycosylated RGY8-6H-Gal2 (1KBG.pdb, in green) (Speir et al., 1999) demonstrates their high conformational similarities. In contrast, the central region of the putative TCR binding site is dominated by the extensive exposure of the tethered carbohydrate (colored in blue), which forms a neoantigen. (C). Glycosylation of MHC-I-restricted peptides can be used by pathogens to either impair TCR recognition of specific epitopes or reduce the overall stability of pMHC complexes. This is exemplified by the crystal structures of the LCMV epitopes GP92 and GP392 in complex with H-2D^b. While glycosylation of the asparagine residue at peptide position 4 in GP92 (5JWE.pdb, in green) may impair TCR recognition, glycosylation of the main anchor asparagine residue at p5 in GP392 (5JWD.pdb, in green) may reduce significantly pMHC stability (Hafstrand et al., 2017).

FIGURE 3

Phosphorylation of MHC-I peptide residues may act as additional external anchor positions, increasing significantly pMHC stability (A). Phosphorylation of the solvent-exposed serine residue at p4 in the HLA-A0201-restricted decameric Lymphocyte specific protein 1-derived peptide (3BH8. pdb) (Mohammed et al., 2008) results in the formation of strong electrostatic interactions between the negatively charged phosphoserine moiety and the surrounding MHC-I residues R66 and K65. Additional interactions are also formed between the phosphorylated moiety and the side chain of peptide residue p1R. Interactions between the phosphate moiety and the basic HLA residues are displayed with yellow dashed lines (B). Similarly, besides forming a hot spot for TCRs, the phosphorylation of the exposed residue in a nonameric peptide derived from the inner centromere protein bound to HLA-B*40:02 (2IEH.pdb) (Alpízar et al., 2017) increases significantly the overall stability of the modified pMHC complexes through the formation of electrostatic interactions with surrounding MHC heavy chain and peptide residues.

FIGURE 4

Citrullination may allow binding of neoantigens to MHC, modify surface electrostatics and alter the conformations of presented peptides (A). The side chain of a citrullinated arginine residue can bind to the slightly positively charged pocket 4 in HLA-DRB1*04:01 while the side chain of an arginine residue will be repelled, as exemplified here by the crystal structure of the citrullinated fibrinogen peptide (6BIL.pdb) (Ting et al., 2018). The peptide, colored in yellow, binds to the cleft of HLA-DRB1*04:01 with the N- and C-termini extending to the left and right of the peptide binding cleft, respectively. The side chain of the citrullinated residue at p4 is indicated, binding to the slightly positively charged pocket 4 in HLA-DRB1*04:01. The surface of HLA-DRB1*04:01 is colored according to surface electrostatics, with negatively and positively charged regions in red and blue, respectively (B). Citrullination of peptides may result in modification of the pMHC surface electrostatic potential, as exemplified by the surfaces of HLA-DRB1*04:01 in complex with the wild-type enolase-derived epitope eno_26-40 (5LAX.pdb, left) or with the citrullinated PTM variant (5JLZ.pdb, right) (Gerstner et al., 2016). Here, citrullination reduces significantly the size of the positively charged region, possibly leading to the selection of alternative TCRs. Both structures are presented from the TCR view (C). The conformation of the self-peptide VIRP_400-408 depends on the MHC-I allele it binds to. Wild-type VIRP_400-408 binds to HLA-B*27:09 with the side chain of the arginine residue p5R protruding towards the solvent and a presumptive TCR (1OGT.pdb, colored in light orange, left panel). Citrullination of p5R in VIRP_400-408 modifies significantly the conformation of the PTM peptide compared to wild-type VIRP_400-408. The side chain of p5R in the citrullinated VIRP_400-408 dives instead within the cleft of B*27:09 forming hydrogen bonds with the side chain of H116 (3B3I.pdb, colored in blue, left panel). VIRP_400-408 takes two different conformations when binding to HLA-B*27:05, one similar to the one described for the HLA-B*27:09/VIRP_400-408 complex and one diametrically different in which the peptide flips and the side chain of p5R forms hydrogen bonds with the side chain of the HLA-B*27:05 aspartate residue D116 (1OGT.pdb, colored in light orange, right panel). Importantly, citrullination of p5R forces the formation of only one peptide conformation in which the side chain of the citrulline residue protrudes towards the solvent (3B6S.pdb, colored in light blue, right panel) (Beltrami et al., 2008).

FIGURE 5

The myristoyl moiety in myristoylated peptides plays an essential role in both MHC-I binding and adequate presentation of the epitope (A). The crystal structure of the rhesus macaque MHC-I molecule Mamu-B*098 in complex with the myristoylated 5-mer lipopeptide derived from SIV Nef protein (4ZFZ.pdb), reveals the dual role of the PTM moiety in both adequate binding to the MHC-I and peptide presentation (Morita and Sugita 2016). (B). The crystal structure of the rhesus macaque MHC-I molecule Mamu-B*05,104 in complex with a N-myristoylated 4-mer lipopeptide derived from the SIV nef protein (6IWG.pdb) shows how the myristoyl moiety occupies the cleft differently (Yamamoto et al., 2019)

FIGURE 6

Proteasomally spliced peptides bind in a canonical mode to MHC molecules (A) Comparison of the conformations of the original KRAS_5-14 peptide (6O53. pdb, cyan) and its cis-spliced variant KRAS_5-6/8–14 (6O4Y.pdb, yellow) both in complex with HLA-A2*01 demonstrates that the splicing results in the formation of a neoepitope through the removal of a central conformational bulge following splicing (Mishto et al., 2019). The red arrow shows the junction between spliced fragments. (B) Although sequentially unrelated, a comparison of the linear nonameric self-peptide 94-LSSPVTKSF-102 derived from immunoglobulin kappa (2RFX.pdb, cyan) (Chessman et al., 2008) and the nonameric cis-peptide LALLTGVRW (6D2T.pdb, yellow) reveals that i) they make use of similar anchor residues (p2A/p2S and pF9/pW9) in order to bind to HLA-B*57:01 (Faridi et al., 2018; Mishto et al., 2019), and ii) that the junction between spliced fragments is protruding towards the solvent, readily available for interactions with TCRs (red arrow) (C). A similar comparison is presented here with two unrelated decameric peptides. The junction between spliced fragments in the trans-spliced epitope TSMSFVPRPW (6D29. pdb, yellow) (Faridi et al., 2018) is also available for interactions with TCRs. The HLA-B*57:01-restricted decameric peptide from small nuclear protein SmD3 54-RVAQLEQVYI-63 (3VRI.pdb) is presented as a reference for a conventional epitope (Illing et al., 2018). The red arrow shows the junction between spliced fragments.

FIGURE 7

Unnatural amino acids can be easily used to assess the importance of peptide residues for pMHC stability and TCR recognition (A) The unnatural aa α-aminobutyrate was used to replace a cysteine residue, abolishing sulfur–π interactions with this peptide residue and the H-2D^b residue Y45 in H-2D^b/Trh4, demonstrating the key role of this type of interaction for the efficient binding of this TEIPP peptide to H-2D^b. Comparison of the crystal structures of H-2D^b in complex with Trh4 (5E8N.pdb) or Thr4-p2ABU (5E8O.pdb) demonstrated that the conformation of the backbone of the altered peptide (green) is identical to wild-type Trh4 (grey) despite significant different overall pMHC stability (Hafstrand et al., 2016) (B) Replacement of two anchor residues in the peptide bound to HLA-B*27:05 (Hülsmeyer et al., 2004) with unnatural amino acids (1JGE.pdb) resulted in the destabilization of MHC/peptide complex and decreased immunogenicity (Jones et al., 2006).

FIGURE 8

Replacement of three residues in the HLA-A*02:01-restricted melanoma epitope Mart-1 with unnatural amino acids did not alter the conformation while enhancing binding affinity.Comparison of the crystal structure of the wild-type Mart-1 peptide ELAGIGILTV (1JF1. pdb, grey) with the APL in which p1E, p2A and p10V were modified to phenylglycine, norvaline and PRG, respectively (4WJ5. pdb, green) (Hoppes et al., 2014). Two HLA-A*02:01 residues that interact with the modified residues in the APL are also indicated.