Structural basis of the CD8 alpha beta/MHC class I interaction: focused recognition orients CD8 beta to a T cell proximal position

Abstract

In the immune system, B cells, dendritic cells, NK cells, and T lymphocytes all respond to signals received via ligand binding to receptors and coreceptors. Although the specificity of T cell recognition is determined by the interaction of T cell receptors with MHC/peptide complexes, the development of T cells in the thymus and their sensitivity to Ag are also dependent on coreceptor molecules CD8 (for MHC class I (MHCI)) and CD4 (for MHCII). The CD8alphabeta heterodimer is a potent coreceptor for T cell activation, but efforts to understand its function fully have been hampered by ignorance of the structural details of its interactions with MHCI. In this study we describe the structure of CD8alphabeta in complex with the murine MHCI molecule H-2D(d) at 2.6 A resolution. The focus of the CD8alphabeta interaction is the acidic loop (residues 222-228) of the alpha3 domain of H-2D(d). The beta subunit occupies a T cell membrane proximal position, defining the relative positions of the CD8alpha and CD8beta subunits. Unlike the CD8alphaalpha homodimer, CD8alphabeta does not contact the MHCI alpha(2)- or beta(2)-microglobulin domains. Movements of the CD8alpha CDR2 and CD8beta CDR1 and CDR2 loops as well as the flexibility of the H-2D(d) CD loop facilitate the monovalent interaction. The structure resolves inconclusive data on the topology of the CD8alphabeta/MHCI interaction, indicates that CD8beta is crucial in orienting the CD8alphabeta heterodimer, provides a framework for understanding the mechanistic role of CD8alphabeta in lymphoid cell signaling, and offers a tangible context for design of structurally altered coreceptors for tumor and viral immunotherapy.

Figure 1

Binding of MHC/peptide complexes to CD8αα and CD8αβ. CD8αα and CD8αβ were coupled to the dextran surface of a CM5 biosensor chip by standard amine coupling chemistry, and increasing concentrations (1μM, 2μM, 5μM, 10μM, 20μM) of either H-2D^d or H-2K^b were sequentially injected over each surface. The zero time point corresponds to the start of the injection of the soluble analyte. Buffer washout was initiated at 240 seconds. Background binding to a mock-coupled surface was subtracted. The peptides bound to H-2D^d and H-2K^b are P18-I10 (RGPGRAFVTI) and IFSK8 (ISFKFDHL) respectively. Calculated K_D values based on a simple monovalent interaction model, AB <--->A + B, were determined from both kinetics and steady-state evaluation of global curve fits using BIAeval 3.2. Data points collected at 5 Hz are plotted in color and corresponding curve fits are in black.

Figure 2

Structure of CD8αβ /H-2D^d/P18I10 complex. Graphics representation of the CD8αβ/H-2D^d/P18I10 complex is shown in ribbon representation (A) colored as H-2D^d heavy chain (green), β2m light chain (magenta), bound P18I10 peptide (light blue), CD8α (orange), and CD8β (slate). Panel (B) shows a rotation about the y axis of about 90°, with the MHC complex in a surface representation, and the CD8αβ heterodimer as ribbons. Panel (C) shows a close-up of the H-2D^d residues that contact CD8. Residues that contact the CD8α chain are shown in orange, and those that contact CD8β are in slate. The single residue, Q226, that contacts both CD8 chains is yellow. Panel (D) is a rotation of (A) of about -90°, with the CD8 heterodimer in a surface representation, and the MHC in ribbons. Panel (E) reveals the residues of CD8 that contact the MHC, colored in green.

Figure 3

Comparison of CD8αβ/H-2D^d complex to other CD8/MHC complexes. Structures of CD8αα/H-2K^b (1BQH) (48), CD8αα/TL (1NEZ) (81), and CD8αα/HLA-A2 (1AKJ) (42) were each superposed on CD8αβ/H-2D^d, and are illustrated here as ribbon diagrams in the same orientation. (A) shows ribbon diagram of CD8αβ/H-2D^d; (B) CD8αα/H-2K^b; (C) CD8αα/TL; (D) CD8αα/HLA-A2. Panel E illustrates the footprint of CD8αβ on H-2D^d/β2m, with the residues contacting CD8β colored blue, those contacting CD8α colored orange, and the single residue (Q226) contacted by both in yellow. Panel F shows the footprint of CD8αα on H-2K^b/β2m, with the residues contacting CD8α1 in blue, those contacting CD8α2 in orange and those contacting both CD8α subunits in yellow.

Figure 4

Close-up examination of residues of the CD8αβ/H-2D^d interface. Panel (A) is a side-by-side stereo view of the intimate interaction of Gln226 of the MHC heavy chain with residues of both chains of CD8. Panel (B) shows Gln226 of H-2D^d and the corresponding density map contoured at 1.5 σ, illustrating the proximity to CD8α Ser37 and CD8β Pro102. Panel (D) shows residues 225 to 227 of H-2D^d of the CD8αβ/H-2D^d complex (green) superposed on the same residues of the unliganded 1.7 Å H-2D^d structure (purple). Movements of the Nε of Gln226 of 3.8 Å and of the Oε1 (4.3 Å) of Glu227 are indicated.

Figure 5

Comparison of bound and free CD8αβ. (A) Coordinates of mouse CD8αβ free (2ATP(49)) were superposed onto the CD8αβ heterodimer in the complex. CD8α and CD8β in the bound complex are orange and slate respectively, and in the unbound state are yellow and pink. Loop designations are shown in a structure based sequence alignment in Fig. S2. (B) The CDR loops, as indicated, are shown in tube representation, and (C) shows CDR loops in the context of the H-2D^d structure. (D) shows an edited close-up of the interactions between CD8β CDR1 Lys27 liganded (slate) and H-2D^d (green). (E) shows the movement of the CD8α CDR2 loop, focused on residue H60.

Figure 6

Structure-based sequence alignment of CD8α, CD8β, and the α3 domain of MHCI. The indicated sequences were aligned as described (83). (A) CD8α alignment. CD8α residues that contact or form hydrogen bonds to MHCI heavy chain are shown as black triangles. Green filled circles are potential N-asparaginyl-glycosylation sites. (B) CD8β alignment. CD8β residues that contact H-2D^d in the CD8αβ/H-2D^d complex are shown as black filled triangles. Green filled circles are potential glycosylation sites. (C) MHCI α3 domain alignment. Indicated residues are: filled circles, H-2D^d α3 domain residues of CD8αβ/H-2D^d that contact CD8α; filled triangles, H-2D^d α3 domain residues of CD8αβ/H-2D^d that contact CD8β; and filled square, H-2D^d α3 domain residue of CD8αβ/H-2D^d that contacts both CD8α and CD8β.

Figure 7

Schematic illustration of distance of CD8αβ from apposed T cell, based on superposition of TCR/MHC complex onto CD8αβ/MHCI complex. Coordinates of the H-2D^d moiety of a P18I10-specific TCR in complex with H-2D^d/β2m/P18I10 determined to 2.0 Å resolution (K.N. and D.H.M., unpublished data) were superposed on the H-2D^d of the CD8αβ/H-2D^d complex using PyMOL (http://www.pymol.org). Stalk regions of CD8α and β as well as transmembrane regions of the indicated chains are shown as dashed lines.