title: 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.
authors: W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
authors: Xin Xiang
authors: XX
authors: Wang
authors: Yili
authors: Y
authors: Li
authors: Yiyuan
authors: Y
authors: Yin
authors: Min
authors: M
authors: Mo
authors: Qian
authors: Q
authors: Wang
authors: Wei
authors: W
authors: Gao
authors: Lili
authors: L
authors: Wang
authors: Roy A
authors: RA
authors: Mariuzza
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015704:CD4 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015798:HLA-DR1 Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D020349:Surface Plasmon Resonance; D015003:Yeasts / Q000235:genetics
chemical_list: D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D015798:HLA-DR1 Antigen; D019151:Peptide Library