Crystal structure of an HIV-binding recombinant fragment of human CD4

Abstract

CD4 glycoprotein on the surface of T cells helps in the immune response and is the receptor for HIV infection. The structure of a soluble fragment of CD4 determined at 2.3 Å resolution reveals that the molecule has two intimately associated immunoglobulin-like domains. Residues implicated in HIV recognition by analysis of mutants and antibody binding are salient features in domain D1. Domain D2 is distinguished by a variation on the β-strand topologies of antibody domains and by an intra-sheet disulphide bridge.CD4, a cell-surface glycoprotein found primarily on T lymphocytes, is required to shape the T-cell repertoire during thymic development and to permit appropriate activation of mature T cells 1 . T cells that recognize antigens associated with class II major histocompatibility complex (MHC) molecules, mainly T helper cells, express CD4. Evidence is accumulating that CD4 and the T-cell receptor coordinately engage class II molecules on antigenpresenting cells to mediate an efficient cellular immune response, and that engaged CD4 may transmit a signal to an associated cytoplasmic tyrosine kinase, p56 lck .CD4 belongs to the immunoglobulin superfamily of molecules which generally serve in recognition processes 2,3 . The sequence of CD4 4,5 indicates that it consists of a large (~370 residues) extracellular segment composed of four tandem immunoglobulin-like domains, a single transmembrane span, and a short (38 residues) C-terminal cytoplasmic tail. The first domain (D1) shares several features with immunoglobulin variable domains, but the sequence similarities between immunoglobulins and the other extracellular domains (D2, D3 and D4) are more remote.In humans, CD4 can be subverted from its normal immuno-supportive role to become the receptor for infection by the human immunodeficiency virus (HIV) 1,6,7 . Recombinant soluble CD4 proteins bind to the HIV envelope glycoprotein gp120, and can thus inhibit viral infection and virus-mediated cell fusion in vitro (refs 8, 9 and references therein). (refs 21-23 and unpublished results), the main flexibility seems to be at the D2 to D3 junction. We have now crystallized a truncated derivative of CD4 that diffracts well, and here we report its atomic structure. This recombinant fragment 8 as secreted from Chinese hamster ovary (CHO) cells consists of residues 1-183 of human CD4 plus two missense residues, Asp-Thr; and it is unglycosylated. This molecule, which we refer to as D1D2, is as active as sCD4 in binding to gp120 (dissociation constant K d ≃ 3 nM) and retains all antibody epitopes mapped to these domains of CD4 (ref. 8 and unpublished results). Others have crystallized similar fragments from the N-terminal half of sCD4 24,25 and the structure of one is reported in the accompanying paper 25 . HHS Public AccessHere we describe the D1D2 structure in comparison with that of immunoglobulin domains, provide a geometrical definition for HIV recognition sites, and discuss implications of the structure for normal CD4 function and evolution of the immunoglobu...

A role for surface hydrophobicity in protein‐protein recognition

“…This cluster is associated with 64.4% of the total accessible surface of the CD4 2-domain fragment, nearly twice that found for the HLA domains. This large fraction of the surface area calculated to be involved in binding is consistent with studies of interactions between CD4 and gp120, monoclonal antibodies, and class I1 MHC molecules 725 (Clayton et al, 1989;Ryu et al, 1990;Wang et al, 1990;Capon &Ward, 1991;Szabo et al, 1992). Limited competition for binding to CD4 is found for these proteins, the exception being some competition between the OKT4 antibody set and the MHC molecule.…”

“…Comparison of CD4 residue mutations (Ryu et al, 1990) affecting gp120 binding to the residues in the calculated protein clusters composing area 111 in Figure 3. Boldface type indicates agreement between predicted and observed residues.…”

The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains

“…A mutant in this region did not affect binding to sCD200. The alternative possibility that the predicted strands are assigned incorrectly is unlikely as they have many of the characteristic residues for example in beta strands B, C and F. Secondly there is little sequence between the two domains suggesting that the domains may be closely linked as found in CD4 domains 1 to 2 and 3 to 4 [16][17][18], respectively, resulting in a relatively rigid protein.…”

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding

“…Other membrane-associated members of the immunoglobulin superfamily (i.e. CD2 and CD4) also typically bind their native ligands at this surface (Ryu et al, 1990;Wang et al, 1990;Jones et al, 1992;Bodian et al, 1994). Opa protein binding to the residues that are involved in the natural function of CEACAM receptors should prevent any adaptive mutations within the host that could abrogate neisserial binding, as such a change would probably also abrogate CEACAM function.…”