HIV-1 gp41 CHR肽CP621-652 N端功能氨基酸的鉴定及其与NHR多肽T21相互作用研究

Identification of functional amino acids of HIV-1 gp41 CHR peptide CP621-652 and interaction with NHR peptide T21

目的确定HIV-1 gp41 CHR肽CP621-652前7个氨基酸的作用以及CP621-652与NHR肽T21复合物的结构取向,为下一步HIV gp41靶点的新型融合抑制剂改造提供线索。方法合成7个丙氨酸(Ala)逐一替换CP621-652前7个氨基酸序列QIWNNMT的突变体,测定其对HIV-1病毒的抑制活性,应用圆二色谱、聚丙烯酰胺凝胶电泳及凝胶排阻测定CP621-652及突变体与gp41 NHR肽T21的相互作用,合成N端带有半胱氨酸的CP621-652、C端带有半胱氨酸的NHR肽T21及相应的同源二聚体,应用二硫键氧化折叠及平衡策略,分析CP621-652与T21复合物的结合取向。结果CP621-652前7个氨基酸的Q^(1)、W^(3)、M^(6)、T^(7)被Ala替换后活性降低,圆二色谱、聚丙烯酰胺凝胶电泳及凝胶排阻色谱显示,CP621-652及突变体与gp41 NHR肽T21存在相互作用,形成螺旋复合物。N端带有半胱氨酸的CP621-652、C端带有半胱氨酸的NHR肽T21折叠后主要形成异源二聚体。交换平衡实验显示,异源二聚体比同源二聚体稳定。结论Q^(1)、W^(3)、M^(6)、T^(7)是CP621-652的功能氨基酸,CP621-652与T21形成反平行螺旋结构。

Objective To determine the functional amino acids of the first seven amino acids of HIV-1 gp41 CHR peptide CP621-652 at N-terminus and the orientation of complex of CP621-652 and NHR peptide T21,and provide a clue for further modifications of novel HIV-1 gp41 fusion inhibitors.Methods Seven CP621-652 variants containing an alanine(Ala)substitution one by one for the first seven amine acids QIWNNMT were synthesized and their inhibitory activities against HIV-1 were determined.The interactions of CP621-652 variants with gp41 NHR-peptide T21 were determined by circular dichroism spectroscopy,electrophoresis and gel exclusion.The variants of CP621-652 and T21 modified by cysteine at N-or C-terminus and their dimers of disulfide bond were synthesized,the binding orientation of the complex of CP621-652 and T21 was analyzed by oxidative folding and equilibrium strategy.Results The replacement of Q^(1),W^(3),M^(6) and T^(7) by Ala resulted in significant decrease in fusion inhibitory activity.The results of circular dichroism spectroscopy,electrophoresis and gel exclusion chromatography showed that CP621-652 and its mutant interacted with T21 to form a helical complex.CP621-652 with cysteine at N-terminal and T21 with cysteine at C-terminal was folded to form a high ratio of heterodimers.The exchange equilibrium experiment showed that the heterodimers were more stable than the homodimers.Conclusion Q^(1),W^(3),M^(6) and T^(7) are the functional amino acids of CP621-652.CP621-652 and T21form antiparallel helical structures.