A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity
A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity
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DOI：
10.1021/bi700666m
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发表时间：
2007-08-14
期刊：
影响因子：
2.9
通讯作者：
Ghirlanda, Giovanna
中科院分区：
文献类型：
--
作者：
Fromme, Raimund;Katiliene, Zivile;Ghirlanda, Giovanna
氰病毒素-N(CV-N)是一种由101个氨基酸组成的蓝藻凝集素，通过与病毒表面膜蛋白GP 120上的N-连接的寡糖苷的高亲和力结合而具有抗HIV活性。该蛋白含有两个碳水化合物结合域A和B，每个结构域在体外都独立地与短寡甘露糖苷结合。与gp120的相互作用可能涉及单个结构域，也可能同时涉及两个结构域；目前尚不清楚哪种模式将产生抗病毒活性。该模型因结构域交换的二聚体形式的形成而变得复杂，其中每个结构域的一部分在两个单体之间交换，其中包含四个功能碳水化合物结合结构域。为了阐明与gp120的多价相互作用是否是抗病毒活性所必需的，我们设计了一个新的突变体P51G-M4-CVN，其中A结构域的结合位点已经被敲除；此外，[P51G]突变阻止了生理条件下结构域交换的二聚体的形成。在这里，我们给出了P51 GM4-CVN的游离型和二甘露糖结合型的1.8埃的晶体结构，揭示了只有结构域B与二甘露糖结合的单体结构。P51G-M4-CVN与gp120的亲和力几乎比wt CV-N低2个数量级，对HIV完全没有活性。在极端条件下制备的P51Gm4-CVN的结构域交换版本中，恢复了与gp120的紧密结合。我们的发现表明，至少有两个油菜素结合位点的存在，无论是通过存在完整的结构域A和B，还是通过形成结构域互换的二聚体，对于活性是必不可少的。
Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp 120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 angstrom of the free and of the dimannose-bound forms of P51 Gm4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51Gm4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two olicomannoside-binding sites, either by the presence of intact domains A and B or by formation of domainswapped dimers, is essential for activity.