The role of charge and multiple faces of the CD8alpha/alpha homodimer in binding to major histocompatibility complex class Imolecules: support for a bivalent model.

Abstract

The CD8 dimer interacts with the a3 domain of major histocompatibility complex class I molecules through two immunoglobulin variable-like domains. In this study a crystal structure-informed mutational analysis has been performed to identify amino acids in the CD8a/a homodimer that are likely to be involved in binding to Recently the crystal structure of a soluble form of the human CD8a/a homodimer was solved (12). In the N-terminal domain of each polypeptide, two sheets consisting of five and four antiparallel (3-strands form a typical immunoglobulin fold. Connecting these strands is a conserved array of loops, three of which are analogous to the complementarity-determining regions of immunoglobulin (CDR1, CDR2, CDR3). Modeling of the electrostatic surface potential of the CD8a/a homodimer has revealed that the membrane-distal, CDR-containing surface of the molecule is predominantly positively charged.The binding site on class I molecules for CD8a/a has been studied in detail. Previous mutational analyses have identifled three clusters of amino acids in the a3 domain that are important for CD8a/a-class I binding and pinpointed one cluster, residues 223-229, as critical for this interaction (1,(13)(14)(15). This cluster is conserved between mouse and human and comprises a highly exposed, negatively charged loop between strands C and D in the a3 domain. In contrast to the body of work that has contributed to the localization of the putative class I binding site for CD8, only one study has focused on defining the complementary interaction site on CD8a/a. Based on the finding that murine CD8a/a did not bind to human HLA class I in a cell-cell adhesion assay, Sanders et al. (16) performed homolog scanning mutagenesis by exchanging human amino acids with their nonconserved murine counterparts. The results ofthis work indicated a role for both the CDR1-like and CDR2-like loops in this speciesspecific interaction. However, since this study focused on the differences between two species, conserved amino acids involved in binding would not have been identified. In addition, the individual amino acids which might be potential contact points could not be determined because these mutants often contained more than one amino acid substitution.In the current study the site of interaction on CD8a/a has been further characterized by using a panel of point mutants generated with the aid of the CD8a/a crystal structure. Analysis of these mutants in a transient cell-cell adhesion assay has identified a core of amino acids that are likely candidates for interaction with class I molecules. Overall, the results support a bivalent model of interaction between one CD8 homodimer and two class I molecules. The role of electrostatic interactions between the predominantly positively charged CD8a/a and the predominantly negatively charged class I a3 domain is also supported by these findings. MATERIALS AND METHODSMutagenesis. Site-directed mutagenesis of the human CD8a cDNA in pBluescript SK(+) was as described (16). To facilitate t...

The Complementarity-Determining Region-Like Loops of CD8α Interact Differently with β2-Microglobulin of the Class I Molecules H-2Kb and Thymic Leukemia Antigen, While Similarly with Their α3 Domains

“…Eighteen to 24 h before transfection, cells were plated out at 2 ϫ 10 5 cells per 60-mm plate. Liposome-mediated transfection was performed, as previously described (13). For expression of the CD8␣␣ homodimer, 4 g of wild-type (WT) or mutant CD8␣ cDNA in 200 l of Optimem was added to 200 l of Optimem plus 16 l of Lipofectamine (both Life Technologies, Grand Island, NY).…”

Production and characterization of transformed B-lymphocytes expressing the membrane defect of Scott syndrome.

“…Human B cell hybridoma were prepared by fusion of EBV-transformed lymphoblasts with the hygromycin B-resistant, hypoxanthine guanine phosphoribosyl transferase (HGPRT) -deficient human myeloma cell line, UC-LUC (22). UC-LUC cells (2 x 107 cells) and EBV-transformed lymphoblasts (4 x I07 cells) were washed twice with serum-free RPMI 1640, and mixed in 50 ml conical tubes.…”

Xenogeneic β2-Microglobulin Substitution Affects Functional Binding of MHC Class I Molecules by CD8+ T Cells

“…A secondary binding site, implicated in coreceptor CD8 engagement, is supplied by a conserved region of the ␣2 domain, the ␣3 domain, and ␤ 2 m (26 -31). Specific influence of m-␤ 2 m on CD8 binding to MHC-I has not been established, although cocrystal studies (19,26), mutational analyses (27)(28)(29), and MHC-I tetramer-binding studies (30,31) suggest that alterations in ␤ 2 m could potentially affect the overall association. Importantly, residues glutamine (Q) 6, threonine (T) 28, Q29, and lysine (K) 58 of m-␤ 2 m have been shown to contact directly with CD8␣ through a series of hydrogen bonds and salt bridges (26).…”