Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA.
Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA.
复制标题
作者：
HIV-1的成熟核衣壳蛋白NCp 7和gag前体中的NC结构域是抗艾滋病药物发现的有吸引力的靶点。NCp 7与茎环3 RNA（SL 3，也称为psi-RNA，在基因组RNA的包装结构域中）的20聚体模拟物的1：1复合物的稳定性受到离子强度变化的强烈影响。NC结构域识别并特异性包装基因组HIV-1 RNA，而静电吸引和高浓度的蛋白质和RNA驱动NCp 7完全包覆成熟病毒体中的RNA。从NCp 7-结合到SL 3的环碱基的特异性相互作用在具有等于或高于0.2 M的[NaCl]的溶液中产生1：1复合物，而静电相互作用可以在等于或低于0.15 M NaCl时占主导地位，导致主要具有1：2 RNA：蛋白质的复合物。1：1和蛋白质过量复合物的持久非平衡混合物可以在这些较低的盐浓度下存在，其中复合物的分布取决于RNA和蛋白质的添加顺序。添加盐导致亚稳态多蛋白质复合物快速重排为1：1。复合物的稳定性也受添加的盐的性质的影响，0.018 M MgCl 2和0.200 M添加的NaCl产生相同的Kd（21 ± 2 nM）;与相同浓度的氯离子相比，乙酸根离子使1：1复合物稳定超过两倍。维持0.2M NaCl或18 mM MgCl 2的盐浓度足以用于实验以区分破坏1：1复合物中的特异性SL 3-NCp 7相互作用的候选药物。
The mature nucleocapsid protein of HIV-1, NCp7, and the NC-domains in gag-precursors are attractive targets for anti-AIDS drug discovery. The stability of the 1:1 complex of NCp7 with a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA) is strongly affected by changes in ionic strength. NC-domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The specific interactions from NCp7-binding to loop bases of SL3 produce 1:1 complexes in solutions that have [NaCl] at or above 0.2 M, while the electrostatic interactions can dominate at and below 0.15 M NaCl, leading to complexes that have mainly 1:2 RNA:protein. Persistent, non-equilibrium mixtures of 1:1 and protein-excess complexes can exist at these lower salt concentrations, where the distribution of complexes depends on the order of addition of RNA and protein. Adding salt causes rapid rearrangement of metastable multi-protein complexes to 1:1. The stability of complexes is also affected by the nature of the added salt, with 0.018 M MgCl2 and 0.200 M added NaCl producing the same Kd (21 ± 2 nM); acetate ion stabilizes the 1:1 complex by more than a factor of two compared to the same concentration of chloride ion. Maintaining a salt concentration of 0.2 M NaCl or 18 mM MgCl2 is sufficient for experiments to distinguish drug candidates that disrupt the specific SL3-NCp7 interactions in the 1:1 complex.