Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein
Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein
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DOI：
10.1073/pnas.96.23.13091
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发表时间：
1999-11-09
影响因子：
11.1
通讯作者：
Gluckman, JC
中科院分区：
文献类型：
--
作者：
Vita, C;Drakopoulou, E;Gluckman, JC
蛋白质-蛋白质相互作用表面通常是大而复杂的，使得这些界面的小模拟物的合理设计成为一个令人生畏的问题。基于CD 4的CDR 2样环和短蝎毒素（scyllatoxin）的β-发夹区域之间的结构相似性，我们将CD 4的9个残基（与HIV-1包膜糖蛋白（gp 120）结合的中心）的侧链转移到蝎毒素支架的结构同源区域。在竞争实验中，得到的27个氨基酸的miniprotein抑制CD 4与gp 120的结合，IC 50为40 μ M。通过NMR的结构分析表明，嵌合β（-）发夹的主链和引入的侧链都采用了与亲本CD 4相似的构象。系统性的单突变表明，大多数CD 4残基从CDR 2样环中复制的微型蛋白，包括关键的Phe-43，进行的结构和功能分析表明，五个额外的突变，一旦纳入微型蛋白，增加其亲和力为100倍的gp 120的IC 50为0.1-1.0 μ M，取决于病毒株。产生的mini-CD 4抑制不同病毒分离株对CD 4（+）细胞的感染。因此，大的蛋白质-蛋白质界面的核心区域可以在miniprotein支架中复制，为开发蛋白质-蛋白质相互作用的抑制剂提供了可能性，这些抑制剂可能代表生物学和药物发现中的有用工具。
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 mu M IC50. 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 IC50 of 0.1-1.0 mu M, 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.