Salt bridge-forming residues positioned over viral peptides presented by MHC class I impacts T-cell recognition in a binding-dependent manner

Abstract

The viral peptides presentation by major histocompatibility complex class I (MHC I) molecules play a pivotal role in T-cell recognition and the subsequent virus clearance. This process is delicately adjusted by the variant residues of MHC I, especially the residues in the peptide binding groove (PBG). In a series of MHC I molecules, a salt bridge is formed above the N-terminus of the peptides. However, the potential impact of the salt bridge on peptide binding and T-cell receptor (TCR) recognition of MHC I, as well as the corresponding molecular basis, are still largely unknown. Herein, we determined the structures of HLA-B*4001 and H-2K^d in which two different types of salt bridges (Arg62-Glu163 or Arg66-Glu163) across the PBG were observed. Although the two salt bridges led to different conformation shifts of both the MHC I α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. Furthermore, through a series of in vitro and in vivo investigations, we found that MHC I mutations that disrupt the salt bridge alleviate peptide binding and can weaken the TCR recognition of MHC I-peptide complexes. Our study may provide key references for understanding MHC I-restricted peptide recognition by T-cells.

Keywords: Crystal structure; MHC class I; Salt bridge; T-cell epitope; TCR recognition; Tetramer.

Graphical abstract

Fig. 1

The impact of MHC I salt bridges on peptide binding capacity. (A) Binding of human hepatitis B virus (HBV) core antigen-derived immunodominant T-cell epitope HBc87-95 to H-2K^d, its three salt bridge mutants (H-2Kd-M1: Mutant R66A, H-2Kd-M2: Mutant E163A and H-2Kd-M3: Mutant R66A&E163A) and three control mutants (H-2Kd-C1:Mutant R157A, H-2Kd-C2:Mutant A158 G and H-2Kd-C3:Mutant S69A) were elucidated by in vitro refolding. The absorbance peak of the H-2K^d complex with the expected molecular mass of 45 kDa was eluted at the estimated volume of 16 mL on a Superdex 200 10/300 G L column. The profile is marked with approximate positions of the molecular mass standards of 75.0, 43.0, and 29.0 kDa. The blue colors in different shades represent the three control mutants, and the yellow, orange, red represent three salt bridge mutants, respectively. (B) Thermostability of peptide HBc87-95 complexed to H-2K^d and its mutants by CD spectroscopy. The T_ms of different complexes are indicated by the temperature when 50% of the protein unfolded at the black dashed line. We repeated CD at least three times for each protein, the results were showed by mean ± SD, Statistical significance of differences between the wild and mutant was determined by student’s t test. **, P < 0.01; ***, P < 0.001. (C) HLA-B*4001, its three salt bridge mutants (B4001-M1: Mutant R62A, B4001-M2: Mutant E163A, and B4001-M3: Mutant R62A&E163A) and three control mutants (B4001-C1: Mutant A158 G, B4001-C2: Mutant R157A and B4001-C3: Mutant I66A) complexed to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N)-derived T-cell epitope N216-225 were refolded in vitro. (D) Thermostability of HLA-B*4001 and its mutants as measured by CD spectroscopy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 2

T-cell recognition of the salt bridge residues of MHC I. (A) Mouse T-cell immune responses induced by human hepatitis B virus (HBV) core antigen-derived T-cell epitope HBc87-95. The Balb/c mice were immunized with HBc87-95 peptide (Peptide group) or PBS (Adjuvant group) together with adjuvants. ELISPOT assays were performed using freshly isolated mouse splenocytes. The non-specific stimulant PHA was used as a positive control, and mock indicates the negative control without any stimulant. (B) Peptide-specific CD8⁺ T-cells stained by tetramers of H-2K^d and mutants. The HBc87-95 peptide-specific CD8⁺ T-cells in the freshly isolated splenocytes from vaccinated mice were stained by H-2K^d tetramer, Kd-M1 (Mutant R66A) tetramer, Kd-M2 (Mutant E163A) tetramer, and Kd-M3 (Mutant R66A&E163A)tetramer, respectively. The hollow dots represent the peptide-immunized group, and the black dots represent the adjuvant group. The control was staining with unrelated tetramer (HLA-A2/influenza peptide GL9). Error bars represent means ± SD. n = 5 mice for per peptide group, n = 3 mice for per adjuvant group. (C) The representative peptide-specific CD8⁺ T-cells stained by tetramers of H-2K^d and mutants. The statistical analysis between two groups used two-way ANOVA with Bonferroni post-tests. ** p <  0.01, *** p <  0.001.

Fig. 3

Two different salt bridges in H-2K^d and HLA-B*4001, as well as other MHC I molecules. Mouse MHC I H-2K^d possesses the Type 1 salt bridge formed by R66 and E163, shown as the vacuum electrostatic surface potential. Mamu-B*17 and Mamu-B*098 form the same type of salt bridge as H-2K^d. Transparent surfaces of the α1 and α2 domains of these three MHC I molecules are shown in orange, while the presented peptides or lipopeptides are shown in yellow sticks. The Type 2 salt bridge is formed by R62 and E163, as in HLA-B*4001, and can also be found in Mamu-A*02, RT1-A1c, HLA-B*2705, HLA-B*4002, and HLA-B*0702. The α1 and α2 domains of these six MHC I molecules are shown in green transparent surfaces, while the peptides are shown in purple sticks. The black dashed lines in the structure represent the hydrogen bonds constructed by residues E163 and R66 (Type 1 salt bridge)/R62 (Type 2 salt bridge). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 4

The interactions between the two residues forming the salt bridge and the adjacent residues of both MHC I and the peptides. The interactions between R66/R62 and E163 that form the salt bridge are denoted as red arrows. The cyan arrows represent the hydrogen bond between E163 and the side chains of the P1 residues of the peptides. The dark blue arrow marks the hydrogen bond between R62 or R66 and the carbonyl group of the P2 residue of the bound peptide in the PBG. In HLA-B*4002, HLA-B*0702 and Mamu-A*02, the cyan dots represent water molecules. The black dashed lines in the structures represent the hydrogen bonds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 5

The different influences on MHC I structural conformation by the two salt bridges. (A) Superimposed structures of MHC I molecules with two different salt bridges. The α1 and α2-helices of H-2K^d, Mamu-B*17, and Mamu-B*098 are shown as yellow, while the α1 and α2-helices of HLA-B*4001, Mamu-A*02, RT1-A1c, HLA-B*4002, HLA-B*0702, and HLA-B*2705 are green. The purple one indicates the different structures of B*2705. R66-E163 of H-2K^d and R62-E163 of HLA-B*4001 are denoted as yellow and green sticks, forming the salt bridge. (B) Using the α1-helix of MHC I as the benchmark, the α2-helix of H-2K^d with the R66-E163 salt bridge (yellow) is shifted ˜1.65 Å compared to the structure of HLA-B*4001 with the R62-E163 salt bridge (green). (C) Conformational difference of peptides in structures of MHC I complexes with R66-E163 and R62-E163 salt-bridges. Compared to peptides under the R62-E163 salt bridge, the residues in the P1 and P2 positions of peptides under the R66-E163 salt bridges have an obvious shift; the arrow represents the direction of displacement. (D and E) The Cα of the P2 residues of the H-2K^d-bound peptides (yellow) are closer to the C-terminus of the PBG compared to the R62-E163 salt bridge (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).