A short HLA-DRA isoform binds the HLA-DR2 heterodimer on the outer domain of the peptide-binding site

Abstract

The human leukocyte antigen (HLA) locus encodes a large group of proteins governing adaptive and innate immune responses. Among them, HLA class II proteins form α/β heterodimers on the membrane of professional antigen-presenting cells (APCs), where they display both, self and pathogen-derived exogenous antigens to CD4⁺ T lymphocytes. We have previously shown that a shorter HLA-DRA isoform (sHLA-DRA) lacking 25 amino acids can be presented onto the cell membrane via binding to canonical HLA-DR2 heterodimers. Here, we employed atomistic molecular dynamics simulations to decipher the binding position of sHLA-DRA and its structural impact on functional regions of the HLA-DR2 molecule. We show that a loop region exposed only in the short isoform (residues R69 to G83) is responsible for binding to the outer domain of the HLA-DR2 peptide-binding site, and experimentally validated the critical role of F76 in mediating such interaction. Additionally, sHLA-DRA allosterically modifies the peptide-binding pocket conformation. In summary, this study unravels key molecular mechanisms underlying sHLA-DRA function, providing important insights into the role of full-length proteins in structural modulation of HLA class II receptors.

Keywords: Antigen presentation; Molecular dynamics; Protein-protein binding; Structural modulation.

Figure 1 -. The initial configuration of the system comprising sHLA-DRA and the HLA heterodimer.

a) Rigid docking predicted the association of the LR_69–83 loop in sHLA-DRA with HLA-DR2 in 70% of the top ten complexes. Only the LR_69–83 loop in sHLA-DRA is displayed for clarity. The LR_69–83 loop is colored based on its status in each predicted complex, i.e. associated with (blue) or dissociated from (red) the HLA-DR2 surface. b) The top view of the top ten complexes shown in a. c) The best-scoring complex from the solvated flexible docking was used to generate the initial configuration of the system. Side-chains of LR_69–83 are shown in the ball-and-stick representation.

Figure 2 -. Two distinct binding modes of sHLA-DRA and HLA-DR2.

a) The nonbonded interaction energy per residue of sHLA-DRA indicated that only the loop region of this molecule strongly associated with HLA-DR2. Standard error bars were computed for the three simulation trials. The heatmap showing the interaction energy of the sHLA- DRA loop region highlights the importance of residues 72, 75, and 76 in HLA-DR2 association. b) Number of contacts at each time step was quantified as the number of HLA-DR2 atoms within 3 Å distance of residues 72, 75, and 76 of sHLA-DRA. c, d, e) shows two distinct modes of sHLA-DRA binding to HLA-DR2. sHLA-DR2 associated with both HLA-DR2 subunits and remained stable throughout the simulation in the first shown binding mode captured in one simulation trial (c). d, e) shows the sHLA-DRA associated only with the outer region of the β-chain, which occurred in two simulation trials. The sHLA-DRA and HLA-DR2 residues are shown in black and royal blue, respectively. The dashed line marks the middle axis of the HLA peptide-binding groove.

Figure 3 -. The key role of F76 in mediating the HLA-DR2 association.

a) depicts sHLA-DRA molecule diffusing away from the HLA-DR2 surface in the F76A mutant using different time frames shown in the left, middle and right panels. b) The distance between the center of masses of mutant sHLA-DRA and HLA-DR2 is shown. c) The surface exposure of sHLA-DRA, wildtype and F76A mutant, in HeLa cells expressing the canonical HLA heterodimers was quantified by ELISA (as described in the Methods section). The levels of the F76A mutant were significantly lower compared to the wildtype molecule as shown in the bar chart (**P ≤ 0.01). d) HeLa cells transfected with either sHLA-DRA or the F76A mutant were tested by Western blotting, using an antibody against their HA tag. Actin levels served as loading controls.

Figure 4 -. The sHLA-DRA molecule allosterically modulates the peptide-binding pockets of HLA-DR2.

a) Binding pockets along the peptide binding groove of HLA-DR2. b) Distributions of peptide binding pocket volumes of HLA-DR2 interacting either with the sHLA-DRA (red), MBP_85–99 peptide (green), or ligand-free state labeled as W/O Ligand (blue). The sHLA-DRA isoform and MBP peptide induced similar fluctuations in most pocket volumes. c) Surface areas of the binding pockets demonstrated less significant changes in response to presence and type of the HLA ligand. The significance levels shown above the boxplots are ANOVA tests per pocket, * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.00005, and ns stands for non-significant.