title: SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Oxford , South Parks Road, Oxford, OX1 3QU, U.K.
authors.forename: Michael
authors.initials: M
authors.lastname: Fairhead
authors.forename: Gianluca
authors.initials: G
authors.lastname: Veggiani
authors.forename: Melissa
authors.initials: M
authors.lastname: Lever
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.forename: Dejan
authors.initials: D
authors.lastname: Mesner
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Omer
authors.initials: O
authors.lastname: Dushek
authors.forename: P Anton
authors.initials: PA
authors.lastname: van der Merwe
authors.forename: Mark
authors.initials: M
authors.lastname: Howarth
mesh_terms: D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D055550:Protein Stability; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010455:Peptides; C549444:traptavidin; D001710:Biotin; D019809:Streptavidin
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