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.
Highlights
To advance bottom-up nanoassembly, it is important to develop building blocks for modular and robust construction, able to withstand real-world challenges.[1]
Traptavidin is an S52G R53D mutant of streptavidin we developed with an ∼10-fold lower off-rate for biotin conjugates, as well as greater thermostability and mechanostability, and is the most stable binder of biotin conjugates.[2,16,27]
Dead streptavidin (D) subunits contain the mutations N23A, S27D, and S45A, which leads to negligible biotin binding but unchanged tetramer stability.[17,27]
Summary
To advance bottom-up nanoassembly, it is important to develop building blocks for modular and robust construction, able to withstand real-world challenges.[1]. Carboxylic acid, HABA), peptides, and nucleic acid aptamers; their binding strengths are all far from the femtomolar stability of biotin binding and these ligands are generally released by competition with biotin.[2,6−11] A unique cysteine can be introduced into streptavidin for chemical labeling,[12,13] but it is limiting to have to work under precise oxidation states for enabling reaction, while new cysteines often impair folding of potential partner proteins To address this challenge we made use of spontaneous amide bond formation, creating a streptavidin tetramer able to form two orthogonal ultrastable interactions. Both SpyTag and SpyCatcher components are genetically encodable, can be positioned at various locations in the protein chain, are reactive under a wide range of conditions, and do not possess cysteines.[14,15] Here we explore how SpyTag and SpyCatcher can be linked to streptavidin components, how these chimeric assemblies enable assembly of complex protein architectures, and how such nanoassemblies can powerfully stimulate cellular signaling
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