Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus.
Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus.
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DOI：
10.1016/j.jmb.2010.06.042
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发表时间：
2010-09-03
影响因子：
5.6
通讯作者：
Yeager M
中科院分区：
文献类型：
--
作者：
Pornillos O;Ganser-Pornillos BK;Banumathi S;Hua Y;Yeager M
HIV-1衣壳被建模为富勒烯锥，其由约250个病毒CA蛋白六聚体和12个CA五聚体组成。CA六聚体的结构一直难以获得，因为六聚体稳定的相互作用是固有的弱，和CA往往自发组装成captain样颗粒。在这里，我们描述了一个两步生化策略，以获得可溶性CA六聚体结晶。首先，通过在相邻亚基的N-末端结构域之间工程化二硫键交联（A14 C/E45 C或A42 C/T54 C）来稳定六聚体。第二，通过突变（W184 A和M185 A）阻止交联的六聚体进一步聚合成超稳定的captain样结构，所述突变干扰连接相邻六聚体的C末端结构域之间的二聚体缔合。两种不同的HIV-1 CA六聚体的结构几乎相同，我们结合了结构的非突变部分，为天然六聚体生成了一个原子分辨率模型。这种用于结构测定的混合方法一般应适用于其他病毒壳粒和蛋白质-蛋白质复合物。
The HIV-1 capsid is modeled as a Fullerene cone that is composed of ~250 hexamers of the viral CA protein and 12 CA pentamers. Structures of CA hexamers have been difficult to obtain because the hexamer-stabilizing interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Here, we describe a two-step biochemical strategy to obtain soluble CA hexamers for crystallization. First, the hexamer was stabilized by engineering disulfide crosslinks (either A14C/E45C or A42C/T54C) between the N-terminal domains of adjacent subunits. Second, the crosslinked hexamers were prevented from polymerizing further into hyperstable capsid-like structures by mutations (W184A and M185A) that interfered with dimeric association between the C-terminal domains that link adjacent hexamers. The structures of two different HIV-1 CA hexamers were nearly identical, and we combined the non-mutated portions of the structures to generate an atomic-resolution model for the native hexamer. This hybrid approach for structure determination should be applicable to other viral capsomers and protein-protein complexes in general.
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影响因子：
5.6
作者：
Barklis, Eric;Alfadhli, Ayna;McQuaw, Carolyn;Yalamuri, Suraj;Still, Amelia;Barklis, Robin Lid;Kukull, Ben;Lopez, Claudia S.
通讯作者：
Lopez, Claudia S.
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
56.9
作者：
Kerfeld, CA;Sawaya, MR;Yeates, TO
通讯作者：
Yeates, TO
影响因子：
5.6
作者：
Lanman, J;Lam, TT;Prevelige, PE
通讯作者：
Prevelige, PE
影响因子：
--
作者：
Huang, HF;Harrison, SC;Verdine, GL
通讯作者：
Verdine, GL