Structurally altered peptides reveal an important role for N-terminal heptad repeat binding and stability in the inhibitory action of HIV-1 peptide DP178
Structurally altered peptides reveal an important role for N-terminal heptad repeat binding and stability in the inhibitory action of HIV-1 peptide DP178
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DOI：
10.1074/jbc.m512475200
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发表时间：
2006-04-07
影响因子：
4.8
通讯作者：
Shai, Y
中科院分区：
文献类型：
--
作者：
Wexler-Cohen, Y;Johnson, BT;Shai, Y
人类免疫缺陷病毒 1 gp41 折叠成六螺旋束，其中三个 C 端七肽重复区域以反平行方式堆积在由三个 N 端七肽重复 (NHR) 形成的卷曲螺旋上。抑制束形成的肽对理解病毒的进入机制做出了重大贡献。 DP178 部分重叠 C 端七肽重复序列，通过未定义的机制阻止束形成；此外，有人建议结合其他 ENV 区域并以未知方式阻止融合。我们使用了两种结构改变的 DP178 肽；在每个中，两个连续的氨基酸被替换成它们的D构型，亲水性N端区域中的D-SQ和疏水性C端区域中的D-LW。重要的是，我们生成了延长的 NHR 肽 N54，获得了 DP178 的完整 N 螺旋对接位点。有趣的是，D-LW 保留了野生型融合抑制活性，而 D-SQ 则表现出显着降低的活性。与抑制数据相关，CD 光谱和荧光研究表明，所有 DP178 肽都与 N54 相互作用，尽管束的稳定性不同。我们得出的结论是，DP178 N 端区域与内源性 NHR 的强结合，而 DP178 C 端序列对核心形成没有显着贡献，对于 DP178 抑制至关重要。表面等离振子共振揭示，C 末端的 D-氨基酸掺入不会影响活性或膜结合，这一发现与 C 末端的额外膜结合位点或膜锚定作用相关，它与 N 末端协同作用以抑制融合。
Human immunodeficiency virus 1 gp41 folds into a six-helix bundle whereby three C-terminal heptad repeat regions pack in an anti-parallel manner against the coiled-coil formed by three N-terminal heptad repeats (NHR). Peptides that inhibit bundle formation contributed significantly to the understanding of the entry mechanism of the virus. DP178, which partially overlaps C-terminal heptad repeats, prevents bundle formation through an undefined mechanism; additionally it has been suggested to bind other ENV regions and arrest fusion in an unknown manner. We used two structurally altered DP178 peptides; in each, two sequential amino acids were substituted into their D configuration, D-SQ in the hydrophilic N-terminal region and D-LW in the hydrophobic C-terminal. Importantly, we generated an elongated NHR peptide, N54, obtaining the full N-helix docking site for DP178. Interestingly, D-LW retained wild type fusion inhibitory activity, whereas D-SQ exhibited significantly reduced activity. In correlation with the inhibitory data, CD spectroscopy and fluorescence studies revealed that all the DP178 peptides interact with N54, albeit with different stabilities of the bundles. We conclude that strong binding of DP178 N-terminal region to the endogenous NHR, without significant contribution of the C-terminal sequence of DP178 to core formation, is vital for DP178 inhibition. The finding that D-amino acid incorporation in the C terminus did not affect activity or membrane binding as revealed by surface plasmon resonance correlates with an additional membrane binding site, or membrane anchoring role, for the C terminus, which works synergistically with the N terminus to inhibit fusion.