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

Protein-protein interacting surfaces are usually large and intricate, makin g the rational design of small mimetics of these interfaces a daunting prob lem. 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 regio n of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 mu M IC50. Structural analysis by NMR showed that both the backbone of the chim eric beta(-)hairpin and the introduced side chains adopted conformations si milar to those of the parent CD4. Systematic single mutations suggested tha t most CD4 residues from the CDR2-like loop were reproduced in the miniprot ein, including the critical Phe-43, The structural and functional analysis performed suggested five additional mutations that, once incorporated in th e miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0 .1-1.0 mu M, depending on viral strains. The resulting mini-CD4 inhibited i nfection of CD4(+) cells by different virus isolates. Thus, core regions of large protein-protein interfaces can be reproduced in miniprotein scaffold s, offering possibilities for the development of inhibitors of protein-prot ein interactions that may represent useful tools in biology and in drug dis covery.