Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4-HLA-DR1 complex

Abstract

Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4-MHC class II interaction is its exceptionally low affinity, which ranges from K(D) = ∼200 μM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell-cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with K(D) = 8.8 μM, compared with >400 μM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and β2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development.

Fig. 1.

Affinity maturation of human CD4 by yeast surface display. (A) The CD4 mutant library was sorted by 1 μM HLA-DR1 tetramer for round 1 (Rd1), 0.1 μM HLA-DR1 tetramer for round 2 (Rd2), and 4 μM HLA-DR1 monomer for round 3 (Rd3). The positive population (boxed) is shown in the gate. (B) Amino acid sequence alignment of wild-type (WT) CD4 and the dominant A1 mutant from Rd3 around the positions targeted for mutagenesis (residues 35, 40, 42–48, 59, 60, and 63). Mutated residues are shaded; only Arg59 is wild type.

Fig. 2.

Reversion mutagenesis of affinity-matured CD4. (A) Dot plots showing labeling of yeast cells displaying wild-type CD4, affinity-matured CD4 A1 mutant, or CD4 A1 reversion mutants by 1.5-μM SAPE-conjugated HLA-DR1 monomer. Each of the 11 mutated residues in CD4 A1 was individually back-mutated to wild type (A1/position/wild type). (B) Fluorescence intensity of each CD4 A1 reversion mutant was normalized by that of the parental CD4 A1 clone and plotted against the mutation.

Fig. 3.

Mutagenesis of wild-type CD4. (A) Dot plots showing binding of HLA-DR1 tetramers to yeast cells displaying wild-type CD4, affinity-matured CD4 A1, or CD4 bearing the mutations Q40Y/T45W (CD4-DM), T45W/P48L, or Q40Y/T45W/P48L. Yeast cells were labeled with SAPE-conjugated HLA-DR1 tetramers (1 μM) and anti–c-myc mAb 9E10, followed by AF488-conjugated goat anti-mouse secondary antibody. The boxed populations show HLA-DR1 tetramer binding. (B) Dot plots showing binding of HLA-DR1 monomers to yeast cells displaying wild-type CD4, CD4 A1, or CD4-DM (Q40Y/T45W) with the mutations K35P, F43L, K46R, G47S, S60R, D63R, or S60R/D63R (CD4-TM). Cells were labeled with SAPE-conjugated HLA-DR1 monomers (4 μM) and anti–c-myc mAb 9E10.

Fig. 4.

Structure of the human CD4–HLA-DR1 complex. (A) CD4 (yellow) contacts both the α2 (cyan) and β2 (blue) domains of the MHC class II molecule through its D1 domain. The HA peptide bound to HLA-DR1 is red. The strands of the β-sheets of the interacting variable Ig-like domains are labeled. (B) The CD4–HLA-DR1 binding interface. The two regions of CD4-DM (region 1: residues 35–48; region 2: residues 59–63) that contact HLA-DR1 are drawn in stick representation with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. The HLA-DR1 molecular surface that interacts with CD4-DM is shown with the α2 domain in cyan and the β2 domain in blue.

Fig. 5.

The CD4–HLA-DR1 binding interface. (A) Close-up view of the interactions between region 1 of affinity-matured CD4-DM (yellow) and the HLA-DR1 β2 domain (blue). The side chains of interacting residues are shown in ball-and-stick representation with carbon atoms in brown (CD4) or green (HLA-DR1), oxygen atoms in red, and nitrogen atoms in blue. The mutated Tyr40 and Trp45 residues of CD4-DM are in magenta. Hydrogen bonds are drawn as dotted black lines. (B) Interactions between region 2 of CD4-DM (yellow) and the HLA-DR1 α1 domain (cyan). (C) Interactions between region 2 of affinity-matured CD4-TM and the HLA-DR1 α1 domain. The mutated Arg60 and Arg63 residues of CD4-TM are in magenta. (D) Sequence alignment of the CD4-contacting regions of the α- and β-chains of different human (DRA*0101/DRB1*0101, DPA1*0104/DPB1*01011, DQA1*01012/DQB1*0401) and mouse (I-A^k, I-E^k) MHC class II alleles. Residues that contact CD4 in the CD4-DM–HLA-DR1 structure are denoted by triangles. White characters on a black background show residues that are strictly conserved across human or mouse MHC class II molecules. The remaining residues are black.