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 = w200 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 atomiclevel 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.T he analysis of leukocyte surface molecules involved in cellcell recognition has been critical to advancing our understanding of immunological phenomena, such as T-cell activation. From the wide range of interactions examined to date, it has emerged that leukocyte surface molecules interact with remarkably low affinities, with dissociation constants (K D 's) generally between 1 and 100 μM, mainly due to fast off-rates (k off >1 s −1 ) (1-3). For example, T-cell receptors (TCRs) bind cognate peptide-MHC (pMHC) complexes with K D 's between 1 and 100 μM, compared with w10 nM for typical antibody-antigen interactions. The low affinities and rapid kinetics (both on-and offrates) of TCR binding to pMHC are believed to allow T cells to scan with high speed and sensitivity the numerous self-pMHC complexes on antigen-presenting cells (APCs) to detect and respond to antigens expressed at low numbers (4).Of all leukocyte cell-cell recognition molecules characterized so far, the T-cell coreceptor CD4 is the most enigmatic in terms of its binding properties (1). The interaction of CD4 with MHC class II molecules greatly augments cytokine production by helper T cells (5) and substantially reduces the number of antigenic peptides on APCs required for T-cell triggering (6). Surprisingly, however, CD4 binds MHC class II with exceptionally low affinity compared with other cell-cell recognition molecules (K D = 1-100 μM) (1-3), including the T-cell coreceptor CD8, which binds MHC class I molecules. For the CD4-MHC class II interaction, K D 's have been vario...

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

“…To confirm that this measurement was reliable we established a binding assay wherein, in a highly multivalent form, sCD4 binding to cell-expressed pMHC II could be detected. Using this assay we confirmed that for native CD4 the binding site on pMHC II corresponds to that suggested by crystal structures of cross-species and affinity-matured CD4/pMHC II complexes (5,6). It can therefore be assumed that native CD4 forms the same "v-shaped" complex that affinity-matured CD4 forms with TCR/pMHC II, wherein contact with the TCR is seemingly precluded (19).…”

“…CD4 comprises two pairs of V-set and C2-set Ig superfamily domains, with early mutational data showing that the "top" two domains bind pMHC II (4). Crystal structures of cross-species and affinitymatured CD4/pMHC II complexes suggest that CD4 binds a pocket formed by the α2 and β2 domains of pMHC II (5,6). The role of coreceptors in heightening T-cell responses is well established.…”

“…The SPR-based affinity of CD8 for pMHC I is 50-200 μM (10). However, SPR has thus far failed to detect interactions between CD4 and pMHC II, setting a lower limit of the K d at least two orders of magnitude higher than for typical interacting leukocyte-expressed proteins (5,10,11).…”

Crystal structure of a complete ternary complex of T-cell receptor, peptide–MHC, and CD4

“…One of these variants, which contains two substitutions in CD4 D1 (Gln40Tyr/ Thr45Trp), was produced as a full-length ectodomain (CD4 D1-D4) by secretion from baculovirus-infected insect cells. This CD4 mutant bound HLA-DR1 with K D = 8.8 μM, compared with no detectable binding for wild-type CD4, even at concentrations up to 400 μM (27). The CD4 mutant exhibited similar affinity for HLA-DR4 (K D = 10.1 μM), as measured by surface plasmon resonance (SPR) (Fig.…”

“…Indeed, the affinity of the pMHC-CD4 interaction, the dissociation constant (K D ) of which has been variously estimated to range from ∼200 μM to >2 mM (13,26), is even weaker than that of most TCR-pMHC interactions (1-100 μM) (13). To overcome this difficulty, we used in vitro-directed evolution by yeast surface display to increase the affinity of human CD4 for the MHC class II molecule HLA-DR1 (27). We subjected the CD4 D1 domain to in vitro random mutagenesis, and displayed the resulting mutant CD4 library on the yeast surface by fusion to agglutinin protein Aga2p.…”