Fromme Raimund, Katiliene Zivile, Giomarelli Barbara, Bogani Federica, Mc Mahon James, Mori Toshiyuki, Fromme Petra, Ghirlanda Giovanna
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Biochemistry. 2007 Aug 14;46(32):9199-207. doi: 10.1021/bi700666m. Epub 2007 Jul 18.
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 gp120. 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 A of the free and of the dimannose-bound forms of P51G-m4-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 P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.
蓝藻素-N（CV-N）是一种由101个氨基酸组成的蓝藻凝集素，对HIV具有强大的抗病毒活性，其作用机制是通过与病毒表面包膜蛋白gp120上的分支N-连接寡甘露糖高亲和力结合来介导的。该蛋白包含两个碳水化合物结合结构域，A和B，每个结构域在体外都能独立结合短寡甘露糖。与gp120的相互作用可能涉及单个结构域或同时涉及两个结构域；目前尚不清楚哪种模式会引发抗病毒活性。该模型因形成结构域交换二聚体形式而变得复杂，在这种形式中，每个结构域的一部分在两个单体之间交换，该二聚体包含四个功能性碳水化合物结合结构域。为了阐明与gp120的多价相互作用对于抗病毒活性是否必要，我们构建了一种新型突变体P51G-m4-CVN，其中结构域A上的结合位点已被敲除；此外，[P51G]突变可防止在生理条件下形成结构域交换二聚体。在此，我们展示了P51G-m4-CVN游离形式和双甘露糖结合形式在1.8埃分辨率下的晶体结构，揭示了一种单体结构，其中只有结构域B与双甘露糖结合。P51G-m4-CVN与gp120的结合亲和力比野生型CV-N低近2个数量级，并且对HIV完全无活性。在极端条件下制备的P51G-m4-CVN结构域交换版本中，与gp120的紧密结合得以恢复。我们的研究结果表明，至少存在两个寡甘露糖结合位点，无论是通过完整的结构域A和B的存在还是通过形成结构域交换二聚体，对于活性都是必不可少的。
