Charge-based interactions through peptide position 4 drive diversity of antigen presentation by human leukocyte antigen class I molecules

Abstract

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8 + T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Non-pocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here we show that two conserved positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to five-fold. The impact of the R65 mutation demonstrates that non-pocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

APE-Gen2.0: Expanding Rapid Class I Peptide–Major Histocompatibility Complex Modeling to Post-Translational Modifications and Noncanonical Peptide Geometries

“…Both the phosphorylated and non-phosphorylated peptides are characterized by a binding affinity value (measured in nM). For the majority of peptides binding to HLA-A*02:01, phosphorylation is seen at position 4, creating a negative charge which improves binding, as previously discussed . A notable exception is the β-catenin peptide (YLDSGIHSGA, PDB codes: 3FQN, 3FQR), where the phosphorylation does not contribute to better binding as expected.…”

“…For the majority of peptides binding to HLA-A*02:01, phosphorylation is seen at position 4, creating a negative charge which improves binding, as previously discussed. 58 A notable exception is the β-catenin peptide (YLDSGIHSGA, PDB codes: 3FQN, 3FQR), 42 where the phosphorylation does not contribute to better binding as expected. decreases binding affinity, as it has been previously observed.…”

“…Additionally, we wanted to check whether APE-Gen2.0 structural models hint at the downstream effects that the PTM might have on the pMHC complex, particularly on binding affinity. We collected a small set of phosphorylated peptides from the IEDB that also come with their non-phosphorylated counterpart, comprising two alleles HLA-A*02:01 and HLA-B*40:02, for which the effects that phosphorylation has in binding affinity are known. ,, The aforementioned phosphorylated/non-phosphorylated pairs were modeled using APE-Gen2.0, using a 5-experiment protocol, where the modeling is repeated 5 different times to enhance robustness (see Methods). After modeling, for each phosphorylated/non-phosphorylated pair, we compare the two values resulting from the aforementioned protocol.…”

Evolution by innovation as a driving force to improve TCR-T therapies

“…HLA proteins encoded by different HLA alleles are designated as HLA allotypes and each individual has multiple MHC class I and class II alleles that encode their different HLA allotypes. Each HLA allotype has a unique peptide binding groove that accommodates different peptides that share some complementary features (46)(47)(48)(49), but each single HLA molecule will bind only one peptide, forming the pHLA complex that can interact with a single TCR (50). Many thousands of different pHLA complexes can be expressed on the surface of a cancer cell, based on the entire proteome of the cell as a source of peptides and the different HLA allotypes of the patient, as revealed by MS studies (8,(46)(47)(48)(49).…”

“…Each HLA allotype has a unique peptide binding groove that accommodates different peptides that share some complementary features (46)(47)(48)(49), but each single HLA molecule will bind only one peptide, forming the pHLA complex that can interact with a single TCR (50). Many thousands of different pHLA complexes can be expressed on the surface of a cancer cell, based on the entire proteome of the cell as a source of peptides and the different HLA allotypes of the patient, as revealed by MS studies (8,(46)(47)(48)(49). Most pHLA complexes will be ignored by T cells due to immune tolerance for peptides originating from normal cellular proteins, so-called self-peptides.…”