Biron Zohar, Khare Sanjay, Quadt Sabine R, Hayek Yehezkiel, Naider Fred, Anglister Jacob
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Biochemistry. 2005 Oct 18;44(41):13602-11. doi: 10.1021/bi0509245.
The HIV-1 envelope glycoprotein gp41 is responsible for viral fusion with the host cell. The fusion process, as well as the full structure of gp41, is not completely understood. One of the strongest inhibitors of HIV-1 fusion is a 36-residue peptide named T-20, gp41(638-673) (Fuzeon, also called Enfuvirtide or DP-178; residues are numbered according to the HXB2 gp160 variant) now used as an anti HIV-1 drug. This peptide also contains the immunogenic sequences that represent the full or partial recognition epitope for the broadly neutralizing human monoclonal antibodies 2F5 and 4E10, respectively. Due to its hydrophobicity, T-20 tends to aggregate at high concentrations in water, and therefore the structure of this molecule in aqueous solution has not been previously determined. We expressed a uniformly 13C/15N-labeled 42-residue peptide NN-T-20-NITN (gp41(636-677)) and used heteronuclear 2D and 3D NMR methods to determine its structure. Due to the additional gp41-native hydrophilic residues, NN-T-20-NITN dissolved in water, enabling for the first time determination of its secondary structure at near physiological conditions. Our results show that the NN-T-20-NITN peptide is composed of a mostly unstructured N-terminal region and a helical region beginning at the center of T-20 and extending toward the C-terminus. The helical region is found under various conditions and has been observed also in a 13-residue peptide gp41(659-671). We suggest that this helical conformation is maintained in most of the different tertiary structures of the gp41 envelope protein that form during the process of viral fusion. Accordingly, an important element of the immunogenicity of gp41 and the inhibitory properties of Fuzeon may be the propensity of specific sequences in these polypeptides to assume helical structures.
HIV-1包膜糖蛋白gp41负责病毒与宿主细胞的融合。融合过程以及gp41的完整结构尚未完全明确。HIV-1融合的最强抑制剂之一是一种名为T-20（gp41(638 - 673)）的36个残基的肽（福泽昂，也称为恩夫韦肽或DP-178；残基编号根据HXB2 gp160变体），现用作抗HIV-1药物。该肽还包含分别代表广泛中和性人单克隆抗体2F5和4E10的全部或部分识别表位的免疫原性序列。由于其疏水性，T-20在水中高浓度时容易聚集，因此该分子在水溶液中的结构此前尚未确定。我们表达了均匀13C/15N标记的42个残基的肽NN-T-20-NITN（gp41(636 - 677)），并使用异核二维和三维核磁共振方法确定其结构。由于额外的gp41天然亲水性残基，NN-T-20-NITN可溶于水，首次能够在接近生理条件下确定其二级结构。我们的结果表明，NN-T-20-NITN肽由大部分无结构的N端区域和从T-20中心开始并向C端延伸的螺旋区域组成。该螺旋区域在各种条件下均能发现，在一个13个残基的肽gp41(659 - 671)中也观察到过。我们认为，这种螺旋构象在病毒融合过程中形成的gp41包膜蛋白的大多数不同三级结构中得以维持。因此，gp41免疫原性和福泽昂抑制特性的一个重要因素可能是这些多肽中特定序列呈现螺旋结构的倾向。
