The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.

中文翻译：

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HIV-1 gp120相互作用的线性肽抑制剂的作用方式。

线性肽12p1（RINNIPWSEAMM）先前是从噬菌体展示库中分离出来的，被发现可抑制HIV-1 gp120与CD4和CCR5替代物mAb 17b的相互作用[Ferrer，M.和Harrison，S.（1999） J.维罗尔 73，5795-5802]。在这项工作中，我们研究了导致这种双重抑制gp120结合的机制。我们发现存在12p1与gp120的直接相互作用，这种相互作用是在化学计量比为1：1的情况下发生的。该肽在IC（50）值分别为1.1和1.6 microM时抑制单体YU2 gp120与sCD4和17b的结合。12p1肽还抑制了这些配体与三聚体包膜糖蛋白的结合，阻止了gp120与天然共受体CCR5的结合，并在体外特异性抑制了HIV-1对靶细胞的感染。在存在或不存在固定浓度的肽的情况下，单体gp120的sCD4饱和度的分析表明，CD4与gp120结合的12p1抑制是由于变构抑制作用而不是竞争性抑制CD4结合。使用一组显示出对12p1抑制作用减弱的gp120突变体，将肽的推定结合位点定位到紧邻CD4结合足迹但与CD4结合足迹可区分的区域。就肽而言，单个12p1残基取代和各种肽截短的影响表明，Trp7的侧链和12p1的其他结构元件对于gp120结合或有效抑制配体与gp120的结合至关重要。最后，12p1无法抑制sCD4与gp120突变体的结合，该突变体被认为类似于CD4诱导的gp120构象。这些结果表明，12p1在结合CD4之前优先结合gp120。肽与gp120的结合限制了与通常对病毒进入至关重要的配体（CD4和CCR5）的相互作用。更重要的是，这些结果表明12p1结合了一个独特的位点，该位点可能被证明是新型CD4-gp120抑制剂的原型靶标。