Oliva Romina, Falcigno Lucia, D'Auria Gabriella, Dettin Monica, Scarinci Claudia, Pasquato Antonella, Di Bello Carlo, Paolillo Livio
Dipartimento di Chimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia, 80126 Napoli, Italy.
Chembiochem. 2003 Aug 4;4(8):727-33. doi: 10.1002/cbic.200200541.
Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.
尽管存在另一个共有序列Lys500-Ala-Lys-Arg503（位点2），但HIV-1包膜糖蛋白gp160的蛋白水解激活是由前蛋白转化酶弗林蛋白酶在序列R508-E-K-R511（位点1）的C末端选择性进行的。基于跨越gp160序列Pro498-Gly516的合成肽p498的溶液结构分析，我们之前提出N端螺旋可能在调节gp160生理切割位点（包含在一个环中）的暴露和可及性方面发挥作用。在此，我们报告了23个残基的肽h-REKR的活性和构象，该肽设计为具有一个大的N端螺旋，随后是gp160的天然序列Arg508-Gly516。h-REKR被弗林蛋白酶高效消化，效率与完整的天然p498相当。在从纯水到98%三氟乙醇的混合物中的圆二色性分析表明，该肽构象中存在大量螺旋结构。通过DYANA和AMBER模拟，从在三氟乙醇/水中收集的NMR数据获得的分子模型，在N端的大片段上确实具有螺旋结构。这样一个长螺旋似乎并不影响包含C末端位点1的序列的环构象，该序列表现出与完整天然p498已经观察到的相同质子化学位移。
