Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob
Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob
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DOI：
10.1128/jvi.01732-07
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发表时间：
2007-12-01
影响因子：
5.4
通讯作者：
Lieber, Andre
中科院分区：
文献类型：
--
作者：
Wang, Hongjie;Liaw, Yen-Chywan;Lieber, Andre
种属B人腺病毒（Ads）通常与免疫功能低下个体的致命疾病相关。最近，物种B Ad（其中大多数使用普遍表达的补体调节蛋白CD 46作为初级附着受体）已经获得了用作基因治疗载体的兴趣。在这项研究中，我们专注于物种B Ad血清型35（Ad 35），其三聚体纤维结结构域结合三个CD 46分子，K-D（平衡解离常数）为15.5 nM。为了研究Ad 35 knob-CD 46相互作用，我们产生了具有随机突变的Ad 35 knob-CD 46表达文库，并针对CD 46结合对其进行筛选。我们确定了四个关键残基（Phe 242，Arg 279，Ser 282和Glu 302），当突变时，消融Ad 35球与CD 46的结合，而不影响球三聚化。在表面等离子体共振和竞争结合研究中验证了所鉴定的残基的功能重要性。为了模拟Ad 35旋钮-CD 46相互作用，我们通过X射线晶体学以2埃分辨率解析Ad 35旋钮结构，并将其覆盖到Ad 11-CD 46相互作用的现有结构上。根据我们的模型，所有鉴定的Ad 35残基都在与CD 46相互作用的区域中，其中一个CD 46分子在两个结单体之间结合。这种相互作用模式可能对CD 46信号传导和Ad 35的细胞内运输具有潜在的影响。我们的研究结果也为更好地表征B类广告和抗病毒药物的设计，以及应用B类广告作为体内和体外基因转移载体的基础。
Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K-D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-angstrom resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.