Identification and structural analysis of residues in the V1 region of CD4 involved in interaction with human immunodeficiency virus envelope glycoprotein gp120 and class II major histocompatibility complex molecules.

Abstract

The human CD4 molecule binds both human immunodeficiency virus envelope protein gpl20 and class H major histocompatibility complex (MHC) molecules. We have studied a series of mutants in the region of amino acids 42-49 of CD4 for their ability to bind gpl20, to interact with class II MHC, to enhance T-cell activation, and to bind a panel of anti-CD4 antibodies. The mutation Q40P (Gln4O -Pro) and the deletion d42-49 were found to disrupt most antibody epitopes in the V1 domain of CD4, suggesting major conformational changes, whereas mutants F43L, G47R, and P48S retained the binding of most of the anti-CD4 antibodies tested. The mutants d42-49, Q40P, F43L, and G47R lost both gpl20 and class II MHC binding as well as the ability to enhance T-cell activation. In contrast, the mutation P48S affected neither gpl20 binding, nor class H MHC binding, nor T-ceUl activation. We conclude that within this region the binding sites for gpl20 and for class II MHC molecules overlap and that amino acids Phe43 and Gly47 comprise an intimate part ofboth binding sites.These observations are consistent with a three-dimensional model of the V1 domain of CD4 that was developed in order to understand the structural basis for binding to CD4.The CD4 molecule is a surface glycoprotein, expressed in T cells, that associates with the class II major histocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (1-3). When the T-cell receptor (TCR) recognizes antigen in association with class II MHC molecules, the CD4 molecule binds the class II MHC molecule, enhancing a sometimes weak immune interaction (1, 2). The CD4 glycoprotein is also the receptor for the human immunodeficiency virus (HIV), binding the viral envelope glycoprotein gpl20 (4).The CD4 molecule is a single-chain 55-kDa protein with a 372-amino acid extracellular domain consisting of four immunoglobulin-like VJ (variable-joining) or CJ (constantjoining) regions, a 23-amino acid transmembrane domain, and a 38-amino acid cytoplasmic domain (5). The VJ-like (V1) domain of CD4 is 35% homologous to immunoglobulin K light-chain variable regions (6). Soluble forms of the V1 domain appear to block HIV infectivity and syncytium formation in vitro (7-9). Furthermore, epitope-loss mutants of CD4 identified amino acids in the area of residues 42-49 as important for syncytium formation (10). Homolog scanning mutational analysis identified a region between residues 42 and 55 that affects gpl20 binding (7,11). In addition, a much broader region was found to be involved in class II MHC binding compared to that seen affecting gp120 (12,13).Here we report a series of epitope-loss mutants of CD4, involving amino acids 42-49. These mutants were expressed in a T-cell hybridoma that is dependent on CD4 for efficient immune function. Binding of a panel of anti-CD4 antibodies was carried out to determine the effect on the conformation of the molecule. The effect of mutations on the binding of gp120 and class II MHC-bearing B cells, and on the ability of the mutant CD4 m...

Human immunodeficiency virus gp120 binding C'C" ridge of CD4 domain 1 is also involved in interaction with class II major histocompatibility complex molecules.

“…2, 65% vs. 0%6 of specific binding). Partial reduction in adhesion of B cells to a CD4 cDNA-transfected hybridoma containing F43L was also observed by others (43). Leucine, like phenylalanine, has a y-branched side chain and therefore is more structurally related to phenylalanine than isoleucine with its P-branched side chain.…”

“…Thus, other parts of domains 1 and 2 are involved in the interaction with class II molecules. Reports by Bowman et al (43) and Fleury et al (29) also showed interference from changes in strands C' and C" of domain 1. These papers demonstrate negative effects of proline substitutions at either position 40 Sites.…”

Studies on the Interaction of T-Cells with Major Histocompatibility Complex Class II Antigens

“…With the resolution of class I1 structure by X-ray crystallography and the observation that class I1 may exist as dimers of the up heterodimers, it was suggested that the Interactions of T-cells with major histocompatibility complex CD4 binding residues of the P 2 domain of one class I1 heterodimer lie in close apposition to the az domain of the other heterodimer [4,741. This raised the possibility that CD4 may stabilize the 'dimer of dimers' and promote the aggregation of TcRs on the surface of the activating T-cell, especially if CD4 itself has a propensity to dimerize [75, 761. It was evident from domain shuffling data [69], CD4 mutagenesis [63,[77][78][79][80][81][82] and antibody binding studies [62, 64, 831 that more than one region within the CD4 molecule is involved in class I1 binding. The data described in this section exploit the species barrier between mouse class I1 molecules and human CD4 [67][68][69]841 to study the role of the a2 domain in T-cell recognition by introducing the a2 domain of murine H-2E into HLA-DR1.…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…As a key receptor on cell membranes, CD4 is known to physically interact with GP120 (427)(428)(429)(430)(431)(432) and Vpu (14,19,433). Due to the flexible nature of HIV-1 Env, the closed and open conformations of Env drive the dynamic binding of Env to CD4, coreceptors, and antibodies (56,434).…”