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, 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.
Vita
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C
Département d'Ingénierie et d'Etudes des Protéines, Commissariat à l'Energie Atomique, Saclay, 91190 Gif-sur-Yvette, France. claudio.vita@cea.fr
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
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D012604:Scorpion Venoms / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D012604:Scorpion Venoms; C056574:leiurotoxin I