Jupp R A, Phylip L H, Mills J S, Le Grice S F, Kay J
Department of Biochemistry, University of Wales College of Cardiff, UK.
FEBS Lett. 1991 Jun 3;283(2):180-4. doi: 10.1016/0014-5793(91)80583-o.
Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-LeuPhe-) and (b) between the p51 and RNase H domain (-PheTyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
突变被引入到连接区的P2和P1位置，(a)连接逆转录酶(RT)和整合酶(IN)(-亮氨酸苯丙氨酸-)，以及(b)在HIV-1多聚蛋白的RT的p66内的p51和核糖核酸酶H结构域之间(-苯丙氨酸酪氨酸-)。在大肠杆菌中表达这些构建体时，监测HIV蛋白酶(PR)的顺式加工。虽然P1中亮氨酸或苯丙氨酸的存在允许在任一连接点快速切割，但β-分支(异亮氨酸)疏水残基的取代基本上消除了水解。相比之下，在这种-疏水*疏水-连接区侧翼的P2位置放置一个β-分支(缬氨酸)残基导致可裂解肽键的有效切割。然而，P2中的甘氨酸消除了切割。讨论了这些发现对于PR特异性、多聚蛋白加工以及用于结晶目的的同二聚体(p51/p51)RT生成的意义。
