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.

Figure 1

Structural comparison of CD4 and the scorpion scyllatoxin. (A) Three-dimensional structure of CD4 D1–D2 domains (Left; PDB code 1cdh; ref. 35) and of scyllatoxin (Right; PDB code 1scy; ref. 22). Backbone traces are in light blue ribbons (27). The 25–64 region of CD4, binding to gp120 (7), is in orange, and the 36–47 CDR2-like loop, corresponding to the C’C“ β-hairpin, is in red; the 18–29 β-hairpin of scyllatoxin, structurally similar to the CDR2-like loop, is also in red. CD4 side chains of the CDR2-like loop and R59, at the end of strand D, transferred to scyllatoxin, appear as red sticks. (B) Sequence alignment of the CDR2-like loop of CD4, scyllatoxin, CD4M3, double-mutant CD4M8, and quintuple-mutant CD4M9. Transferred amino acid residues of CD4 and amino acid changes of the engineered scaffolds are in blue; the additional changes that increase affinity for gp120 and antiviral activity are in red.

Figure 2

Stereo-view of NMR structure ensemble of CD4M3; 20 models are shown superimposed by using the backbone atoms (in black); disulfide bridges are yellow, and other side chains are blue. Critical side chains are labeled.

Figure 3

Comparison of the 35–48 β-hairpin structure of CD4 with the corresponding 16–27 β-hairpin of CD4M3. Note the good superposition of CD4 backbone structure (green) with that of CD4M3 (magenta) and the similar conformation of the CD4 side chains Gln-40, Phe-43, and Thr-45 (blue) with the corresponding side-chains of CD4M3 (red).

Figure 4

Effect of CD4M9 on gp120-CD4 interaction. Inhibition of gp120_LAI-binding to coated sCD4 in the ELISA. sCD4 (○), CD4M3 (■), double-mutant CD4M8 (▴), and quintuple mutant CD4M9 (●); control peptides are scyllatoxin (▵), linear 37–53 (□), and cyclic 37–46 CD4 peptides (○).

Figure 5

Effect of CD4M9 on HIV-1 infection. (A) Inhibition of HeLa/CD4/CCR5/LacZ cell infection by HIV-1. Closed symbols, CD4M9; open symbols, control sCD4. X4 (LAI, ●, ○) and R5 (BaL, ▴, ▵; Ada, ■, □) HIV-1 strains were tested. Data from one of three experiments are presented as percentage of inhibition of infection. (B) Inhibition of PBL infection by HIV-1. Closed symbols, CD4M9; open symbols, sCD4. The strains were LAI (●, ○) and BaL (▴, ▵). Data from one of three experiments are presented as percentage of inhibition determined on day 10 after infection.