Coadou Gaël, Evrard-Todeschi Nathalie, Gharbi-Benarous Josyane, Benarous Richard, Girault Jean Pierre
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), Université René Descartes-Paris V, 45 rue des Saint-Pères, 75270 Paris, Cedex 06, France.
Int J Biol Macromol. 2002 Mar 8;30(1):23-40. doi: 10.1016/s0141-8130(01)00184-2.
Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.
由HIV-1蛋白Vpu介导的蛋白酶体对HIV受体CD4的降解，对于释放完全具有感染性的病毒粒子至关重要。为促进CD4降解，Vpu必须在基序DSGXXS上磷酸化，该基序在几种已知经磷酸化后会被蛋白酶体降解的信号蛋白中保守。这种磷酸化是Vpu与泛素连接酶SCF-β-TrCP相互作用所必需的，而SCF-β-TrCP会触发蛋白酶体对CD4的降解。在本研究中，我们使用了Vpu第41至62位残基之间的两个22个氨基酸的肽段。Vpu41-62预计会形成一个α-螺旋-柔性-α-螺旋，包括磷酸化基序DS52GNES56，且Vpu_P41-62在Ser52和Ser56这两个位点被磷酸化。我们分析了该肽段在Ser52和Ser56位点磷酸化所诱导的构象变化。利用同核和异核NMR技术评估磷酸化的结构影响。通过联合应用几种二维质子NMR方法，完全归属了游离肽段、Vpu_P41-62和Vpu41-62在H2O（pH 3.5和7.2）以及H2O与三氟乙醇的1:1混合物中的谱图。对短程和中程NOE连接性以及二级化学位移的分析表明，肽段（42-49）的螺旋倾向不太明确。Vpu_P41-62的50-62位残基结构域形成一个环，磷酸基团向外，还有一个短β-链以及60-62位残基的柔性延伸“尾巴”。通过联合（温度、溶剂和pH）NMR和分子计算实验，50-60位残基的α-质子NMR二级化学位移从无规卷曲向更类似β-结构发生变化。50-62这个分子区域的差异表明，Vpu_P的构象变化在Vpu_P诱导的CD4分子降解中起重要作用。
