Fairhead Michael, Veggiani Gianluca, Lever Melissa, Yan Jun, Mesner Dejan, Robinson Carol V, Dushek Omer, van der Merwe P Anton, Howarth Mark
Department of Biochemistry, University of Oxford , South Parks Road, Oxford, OX1 3QU, U.K.
J Am Chem Soc. 2014 Sep 3;136(35):12355-63. doi: 10.1021/ja505584f. Epub 2014 Aug 21.
The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ≤ n ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:β2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.
链霉亲和素对生物素的捕获是超分子化学的灵感来源，也是生物化学和纳米技术的核心工具，这是因为它们之间的相互作用迅速且异常稳定。然而，对于这个核心枢纽，目前还没有强大的正交相互作用，这限制了可以构建的分子组装体的大小和复杂性。在这里，我们将捕鸟亲和素（一种使生物素结合强度最大化的链霉亲和素变体）与一种正交不可逆相互作用相结合。SpyTag是一种经过工程改造的肽，可与其蛋白质伴侣SpyCatcher形成自发的异肽键。SpyTag或SpyCatcher成功地融合到了失活链霉亲和素亚基的C末端。我们能够生成具有n（0≤n≤4）个生物素结合位点和4 - n个SpyTag或SpyCatcher结合位点的嵌合四聚体。嵌合的SpyAvidin四聚体能够结合与生物素或SpyTag/SpyCatcher融合的精确数量的配体。混合嵌合四聚体能够组装成SpyAvidin八聚体（8个亚基）或二十聚体（20个亚基）。我们使用电泳和原生质谱对组装体进行了验证。二十聚体SpyAvidin用于聚集三聚体主要组织相容性复合体（MHC）I类：β2 - 微球蛋白：肽复合物，生成一个包含多达56个组分的组装体。MHC二十聚体在作为T细胞信号传导的强大刺激物方面超过了传统的MHC四聚体。将超稳定的非共价相互作用与不可逆的共价相互作用相结合，SpyAvidin为创建强大的纳米结构提供了一条简单的途径。
