Department of Chemistry, Syracuse University, Syracuse, New York 13244, USA.
Biochemistry. 2010 May 4;49(17):3525-33. doi: 10.1021/bi901279e.
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 of NCp7 binding to loop bases of SL3 produce 1:1 complexes in solutions that have a NaCl concentration of >or=0.2 M, while the electrostatic interactions can dominate at <or=0.15 M NaCl, leading to complexes that have a mainly 1:2 RNA:protein ratio. Persistent, nonequilibrium 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 multiprotein complexes to a 1:1 ratio. The stability of complexes is also affected by the nature of the added salt, with 0.018 M MgCl(2) and added 0.200 M NaCl producing the same K(d) (21 +/- 2 nM); acetate ion stabilizes the 1:1 complex by a factor of more than 2 compared to the same concentration of chloride ion. Maintaining a salt concentration of 0.2 M NaCl or 18 mM MgCl(2) is sufficient for experiments to distinguish drug candidates that disrupt the specific SL3-NCp7 interactions in the 1:1 complex.
HIV-1 的成熟核衣壳蛋白 NCp7 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标。NCp7 与 20 聚体模拟物（基因组 RNA 包装域中的茎环 3 RNA，也称为 psi-RNA）的 1:1 复合物的稳定性强烈受离子强度变化的影响。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 NCp7 完全覆盖成熟病毒粒子中的 RNA。NCp7 与 SL3 环碱基的特异性相互作用在 NaCl 浓度> = 0.2 M 的溶液中产生 1:1 复合物，而静电相互作用可以在< = 0.15 M NaCl 时占主导地位，导致具有主要 1:2 RNA:蛋白比的复合物。在这些低盐浓度下，可以存在 1:1 和蛋白质过量复合物的持久非平衡混合物，其中复合物的分布取决于 RNA 和蛋白质的添加顺序。添加盐会导致亚稳态多蛋白复合物迅速重排为 1:1 比例。复合物的稳定性也受添加盐的性质影响，0.018 M MgCl(2)和添加的 0.200 M NaCl 产生相同的 K(d)（21 +/- 2 nM）; 与相同浓度的氯离子相比，醋酸根离子将 1:1 复合物稳定了 2 倍以上。维持 0.2 M NaCl 或 18 mM MgCl(2)的盐浓度足以进行实验，以区分破坏 1:1 复合物中特异性 SL3-NCp7 相互作用的候选药物。
