Electrostatic interactions drive membrane association of the human immunodeficiency virus type 1 Gag MA domain
Electrostatic interactions drive membrane association of the human immunodeficiency virus type 1 Gag MA domain
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DOI：
10.1128/jvi.02757-06
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发表时间：
2007-06-01
影响因子：
5.4
通讯作者：
Vogt, Volker M.
中科院分区：
文献类型：
--
作者：
Dalton, Amanda K.;Ako-Adjei, Danso;Vogt, Volker M.
大多数逆转录病毒的组装发生在质膜上。膜结合是由Gag结构蛋白的n端结构域MA引导的。对于人类免疫缺陷病毒1型（HIV-1），这种关联部分是由肉豆蔻酸脂肪酸修饰介导的。关于肉豆蔻酰化的相对重要性，蛋白质和膜之间的离子相互作用，以及在体内膜结合中的Gag多聚化，存在着相互矛盾的证据。我们通过测定纯化的肉豆荚酰化HIV-1 MA对确定组成的脂质体的亲和力来解决这些问题，包括对蛋白质的单体和二聚体形式。肉豆蔻酰基化使载脂蛋白对脂质体的内在亲和力仅提高了10倍，并且产生的亲和力仍然很弱，类似于劳斯肉瘤病毒天然未肉豆蔻酰基化的MA。HIV-1 MA的膜结合完全依赖于带负电荷的脂质存在，并在高离子强度下被废除。MA的强制二聚化使其膜亲和性提高了几个数量级。当细胞中表达单体或二聚体的绿色荧光蛋白融合物时，二聚体而非单体蛋白变得与膜紧密相关。计算模型支持这些结果，并提出了肉豆蔻酸基化对结合的适度影响的分子机制，其中膜为肉豆蔻酸提供了一个与蛋白质提供的能量相似的疏水环境。总的来说，结果表明膜结合的驱动力主要来自多聚Gag和带负电荷的磷脂之间的离子相互作用。
The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by, only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.