Structure of a covalently stabilized complex of a human alphabeta T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1

Abstract

An alphabeta T-cell receptor (alphabetaTCR)/hemagglutinin (HA) peptide/human leukocyte antigen (HLA)-DR1 complex was stabilized by flexibly linking the HA peptide with the human HA1.7 alphabetaTCR, to increase the local concentration of the interacting proteins once the peptide has been loaded onto the major histocompatibility complex (MHC) molecule. The structure of the complex, determined by X-ray crystallography, has a binding mode similar to that of the human B7 alphabetaTCR on a pMHCI molecule. Twelve of the 15 MHC residues contacted are at the same positions observed earlier in class I MHC/peptide/TCR complexes. One contact, to an MHC loop outside the peptide-binding site, is conserved and specific to pMHCII complexes. TCR gene usage in the response to HA/HLA-DR appears to conserve charged interactions between three lysines of the peptide and acidic residues on the TCR.

Fig. 1. Linking of the HA306–318 antigen peptide to the TCR β-chain leads to an SDS-stable αβTCR/pMHCII complex. (A) The HA peptide (magenta) was linked via an octapeptide linker (black) to the N-terminus of the Vβ-chain (blue) of the HA1.7 αβTCR (yellow, blue). HA of the resulting p-TCR, HA-HA1.7, was loaded on empty HLA-DR1 (red, green) with HLA-DM. (B) SDS–PAGE (8–16%) of the HA/HA1.7/DR1 complex. Pre-formed HA-HA1.7/DR1 complex was boiled and reduced, just boiled or loaded directly onto the SDS–polyacrylamide gel. Note that the non-boiled and non-reduced HA-HA1.7/DR1 complex does not dissociate on SDS–PAGE, in contrast to the unlinked complex between HA1.7 and DR1/HA. (C) Native gel band-shift assay shows a smear for the HA1.7 TCR/HA/DR1 complex and a distinct band for the HA-HA1.7-linked TCR/DR1 complex. (D) The structure of the p-TCR, HA-HA1.7/DR1 complex with the TCR at the top (yellow, blue) and DR1 at the bottom (red, green). The distance of 19 Å between the C-terminus of the HA peptide (magenta) and residue 3 of the TCR Vβ domain is indicated by an arrow. The figure was created with MOLSCRIPT (Kraulis, 1991) and Raster3D (Merritt and Murphy, 1994).

Fig. 2. Binding of TCRs to MHC class II and class I molecules. Data from five αβTCR/peptide/MHC structures are compared: HA1.7/HA/DR1 (this study), D10/CA/I-A^k (Reinherz et al., 1999), A6/TAX/HLA-A2 (Garboczi et al., 1996a), B7/TAX/HLA-A2 (Ding et al., 1998) and 2C/DEV8/H-2K^b (Garcia et al., 1998). (A) Red MHC residues are contacted by the TCR in the class II and class I complexes named. Yellow underlined sequences correspond to the ‘top’ α-helices and blue underlined sequences to the ‘bottom’ α-helices of the peptide-binding grooves as shown in the ribbon diagrams. Residues of MHCII on the loop between β-strands 3 and 4 are underlined in magenta, and those that precede the N-terminal of the ‘top’ α-helix in green (also on the ribbon diagrams). (B) Orientations of the antigen-combining sites of TCRs with the peptides to define a horizontal axis (Vα is at top right, Vβ is at bottom left, the peptide N-terminus is at the left and the C-terminus is at the right). Positive and negative electrostatic surface potentials of the TCRs are indicated in blue and red, respectively. Figures were prepared with MOLSCRIPT (Kraulis, 1991), Raster3D (Merritt and Murphy, 1994) and GRASP (Nicholls et al., 1991).

Fig. 3. Interaction of TCR HA1.7 with HLA-DR1 and HA peptide. (A) TCR contacts. Contacts of CDR residues (solid red lines) with HLA-DR1 (solid blue lines) and the HA peptide (solid black line) are indicated by dashed green and dashed red lines, respectively. Human MHC class II conserved (filled circles) and polymorphic residues (open circles) are shown. (B) Contacts between CDR2β and the conserved Lys39 of DR1α outside of the peptide-binding site (van der Waals contacts, dashed black lines; potential hydrogen bonds, dashed red lines). (C) MHC–peptide solvent-accessible surface buried by the TCR, colored by CDR type (see key). The total accessible surfaces buried on pMHC by the TCR are 1111 Å² for HA1.7/DR1/HA, 1041 Å² for D10/I-Ak/CA, 1031 Å² for A6/A2/TAX, 918 Å² for B7/A2/TAX and 1111 Å² for 2C/H-2Kb/DEV8. The antigenic peptide is shown by a white line. Figures were prepared with MOLSCRIPT (Kraulis, 1991), Raster3D (Merritt and Murphy, 1994) and GRASP (Nicholls et al., 1991).

Fig. 4. Recognition of the HA peptide by TCR HA1.7. (A) Binding of the HA peptide (yellow) to the surface of the TCR HA1.7 (top) and in the groove of DR1 (bottom). HA1.7 and DR1 were moved apart and rotated around the long axis of the peptide by –20° and +20°, respectively, in order to allow a better view into the peptide-binding sites. Positive and negative electrostatic surface potentials of HA1.7 and DR1 are indicated in blue and red, respectively. (B) van der Waals contacts and potential hydrogen bonds between TCR HA1.7 and HA peptide are shown by black and red dashed lines, respectively. (C) Electrostatic interactions between the three lysines (P–1, P3 and P8) of HA with acidic residues of HA1.7 TCR. (D) HA and CA peptide residues that are contacted by TCR HA1.7 and D10, respectively, are shown in red. The number of peptide residues that are contacted by the different TCRs and the range over which they are distributed are indicated. (A–C) were prepared with MOLSCRIPT (Kraulis, 1991), Raster3D (Merritt and Murphy, 1994) and GRASP (Nicholls et al., 1991).

Fig. 5. Stereographic diagram of the α-carbon paths of five αβTCR/pMHC complexes. (A) Complexes were superimposed on their peptide-binding domains (center). The similar orientations of the TCRs (top) on the pMHC (bottom) are evident when viewed in stereo. (B) As in (A), but rotated by 90° about the vertical axis. TCRs (top) all fit at an angle between high points on the MHC surfaces, when viewed in stereo. Arrows show the difference locations of the CD4 and CD8 binding sites. Complexes shown are as in Figure 2.