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Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.

ABSTRACT: Protein-protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the beta-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 microM IC(50). Structural analysis by NMR showed that both the backbone of the chimeric beta-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC(50) of 0.1-1.0 microM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4(+) cells by different virus isolates. Thus, core regions of large protein-protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein-protein interactions that may represent useful tools in biology and in drug discovery.

SUBMITTER: Vita C

PROVIDER: S-EPMC23905 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.

Vita C C Drakopoulou E E Vizzavona J J Rochette S S Martin L L Ménez A A Roumestand C C Yang Y S YS Ylisastigui L L Benjouad A A Gluckman J C JC

Proceedings of the National Academy of Sciences of the United States of America 19991101 23

Protein-protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the beta-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition e ...[more]

PMID: 10557278

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Project description:UnlabelledThe HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5.ImportancePotent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that, unlike soluble forms of CD4, CD4bs antibodies poorly induce envelope glycoprotein conformations that efficiently bind CCR5. This study provides insight into the properties of potent CD4bs antibodies and suggests that, under some conditions, CD4i antibodies can improve their potency. These observations may be helpful to the development of vaccines designed to elicit specific antibody classes.

Dataset Information

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.

Publications

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.

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