Structural characterisation of cd81-claudin1 hepatitis c cirus receptor complex

约翰逊和约翰逊（J&J）投资于五个治疗领域，这些领域仍然存在重大的新药开发需求。其中之一是抗病毒疗法，现在人们认识到迫切需要设计抑制病毒感染早期步骤的药物。这与当前一代的蛋白酶和聚合酶抑制剂形成对比，后者在临床上快速选择病毒变体。事实上，这些变异体在治疗受试者中出现的速度表明，丙型肝炎病毒（HCV）等病毒正在感染新的细胞。拟议的合作解决了该公司在开发针对丙型肝炎病毒感染等传染病的新药方面的战略利益。通过了解关键受体分子的结构和细胞生物学，该项目将产生新的发现管道。我们建议，结构和功能表征的两个膜蛋白，每个具有4个跨膜结构域，CD 81和Claudin-1（CLDN 1），将使其蛋白质-蛋白质相互作用的机制得到阐明。CLDN 1和CD 81形成使HCV能够感染细胞的受体。这两种共受体组分的寡聚化状态对该过程至关重要。我们已经表明，CD 81在质膜上寡聚化，鉴定出CD 81-CD 81同源二聚体和CD 81-CLDN 1异源二聚体，表明CLDN 1募集到富含CD 81的结构域。CD 81-CLDN 1复合物的扰动抑制HCV进入，表明在病毒进入过程中起关键作用。此外，我们最近已经鉴定了CLDN 1的第一胞外环（EC 1）中的氨基酸残基，其定义了与CD 81和病毒共受体活性的关联。比尔实验室通过提供合适的重组膜蛋白靶点和不同寡聚状态的突变体来做出贡献。McKeating实验室为这些蛋白质的生物测定提供专业知识，以及获得一系列独特的抗体试剂。比尔和麦基廷以前曾一起出版过，并有一些额外的手稿在准备中。了解这些分子将有助于设计针对病毒感染的治疗药物。我们的具体目标将解决以下问题：A。CD 81的寡聚状态是什么？迄今为止，由于缺乏适当的工具，CD 81及其家族成员（四跨膜蛋白）的寡聚状态在很大程度上是未知的，蛋白质构象和生物活性之间的关系也很难确定。我们假设HCV使用二聚体CD 81进入细胞。我们将测试这一假设，并确定蛋白质基序定义四跨膜蛋白寡聚化和受体活性。我们正在进行的单体和寡聚体CD 81的结晶试验（我们有单体CD 81的衍射晶体）将补充这项工作，因为没有任何一个家族的全长成员的结构信息。B。CLDN 1的寡聚状态是什么？我们最近报道了基于标记分子之间的荧光共振能量转移（FRET）的CLDN 1二聚化，表明二聚体是紧密连接链的主要构建块。生物物理和结构表征将确定HCV使用的寡聚体状态和构象。C. CD 81-CLDN 1共受体复合物的结构组织是什么？我们最近证明酵母表达的CD 81和CLDN 1可以结合HCV。酵母没有CD 81或CLDN 1同源物，这使我们能够研究（而不是间接地在哺乳动物细胞中）CD 81和CLDN 1结合的结构基础和病毒共受体活性。在药物发现管道的背景下，这种膜蛋白生物化学和细胞生物学的合作研究计划为所有合作伙伴，特别是博士生提供了一个退出的机会。

Johnson & Johnson (J&J) invests in five therapeutic areas where there continues to be significant unmet need for novel drug development. One of these is anti-viral therapies, where it is now recognised that there is an urgency to design drugs that inhibit the early steps of viral infection. This contrasts with the current generation of protease and polymerase inhibitors which rapidly select viral variants in the clinic. Indeed, the rapidity at which these variants appear in treated subjects suggests viruses such as hepatitis C virus (HCV) are infecting new cells. The proposed collaboration addresses the company's strategic interest in developing new drugs targeted at infectious diseases such as HCV infections. By gaining an understanding of the structural and cell biology of key receptor molecules, novel discovery pipelines will emerge from this project. We propose that structural and functional characterisation of two membrane proteins each with 4 transmembrane domains, CD81 and Claudin-1 (CLDN1), will enable the mechanism underlying their protein-protein interactions to be elucidated. CLDN1 and CD81 form a receptor that enables HCV to infect cells. The oligomerisation status of these two co-receptor components is critical to this process. We have shown that CD81 oligomerises at the plasma membrane, with the identification of CD81-CD81 homodimers and CD81-CLDN1 heterodimers suggesting a recruitment of CLDN1 to CD81-enriched domains. Perturbation of CD81-CLDN1 complexes inhibits HCV entry, suggesting a critical role in the viral entry process. Furthermore, we have recently identified the amino acid residues in the first extracellular loop (EC1) of CLDN1 that define association with CD81 and viral co-receptor activity. The Bill laboratory contributes by providing access to suitable recombinant membrane protein targets and mutants of varying oligomeric states. The McKeating laboratory provides expertise in biological assays for these proteins, as well as having access to a range of unique antibody reagents. Bill and McKeating have previously published together, and have a number of additional manuscripts in preparation. Understanding these molecules will aid the design of therapeutic agents targeting viral infection. Our specific aims will address the following questions: A. What is the oligomeric status of CD81? To date, because of the lack of appropriate tools, the oligomeric status of CD81 and its family members (the tetraspanins) is largely unknown and the relationship between protein conformation and biological activity is poorly defined. We hypothesise that HCV uses dimeric CD81 to enter cells. We will test this hypothesis and identify protein motifs defining tetraspanin oligomerisation and receptor activity. Our ongoing crystallisation trials of monomeric and oligomeric CD81 (we have diffracting crystals of monomeric CD81) will complement this work, since there is no structural information available for any full-length member of either family. B. What is the oligomeric status of CLDN1? We recently reported CLDN1 dimerisation based on fluorescence resonance energy transfer (FRET) between tagged molecules, suggesting that dimers are the primary building block(s) of tight junction strands. Biophysical and structural characterisation will identify the oligomeric status and conformation used by HCV. C. What is the structural organisation of the CD81-CLDN1 co-receptor complex? We recently demonstrated that yeast-expressed CD81 and CLDN1 can bind HCV. Yeast does not have CD81 or CLDN1 homologues, allowing us to study (rather than indirectly in mammalian cells) the structural basis of CD81 and CLDN1 association and viral co-receptor activity. This collaborative research programme in membrane protein biochemistry and cell biology, in the context of the drug discovery pipeline, provides an exiting opportunity for all partners and especially a PhD student.