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.

Figure 1

Nanohubs with biotin binding and SpyTag/SpyCatcher ligation. (A) Multiple noncovalent interactions in biotin binding. The tight binding of biotin (carbons in yellow) by traptavidin from the formation of eight hydrogen bonds as well as a hydrophobic cage of tryptophans, with residues mutated in traptavidin in cyan (from PDB 2Y3F). (B) Spontaneous isopeptide bond formation leads to a covalent linkage between a SpyTag-labeled protein target (cyan) and SpyCatcher (dark blue) (from PDB 4MLS). (C) Combining SpyTag/SpyCatcher with traptavidin/biotin allows the formation of tetramers with subunits able to bind biotin (Tr or Tre), bind SpyTag (DCatch), or bind SpyCatcher (DTag).

Figure 2

Generation of traptavidin tetramers linked to SpyTag. (A) Formation of chimeric tetramers of traptavidin-glutamate tag (Tre) and Dead streptavidin-SpyTag (DTag) by mixed refolding of inclusion bodies and separation by ion-exchange chromatography. Predicted pI values are shown for the constituent monomers and the assembled tetramers. (B) Ion-exchange chromatogram showing separation of the mixed refold into individual species bearing different numbers of functional biotin binding sites and SpyTags. A₂₈₀ is plotted with a solid line, and conductivity, with a dotted line. (C) SDS-PAGE with Coomassie staining of Tre/DTag tetramers showing the tetramer mobilities in unboiled samples (left) and the subunit compositions in boiled samples (right). Mix refers to the input sample.

Figure 3

Generation of traptavidin tetramers linked to SpyCatcher. (A) Formation of chimeric tetramer forms of traptavidin (Tr) and Dead streptavidin-SpyCatcher (DCatch) by mixed refolding of inclusion bodies and separation by ion-exchange chromatography. Predicted pI values are shown for the constituent monomers and the assembled tetramers. (B) Ion-exchange chromatogram showing separation of the mixed refold into individual species bearing different numbers of functional biotin binding sites and SpyCatchers. A₂₈₀ is plotted with a solid line, and conductivity, with a dotted line. (C) 14% SDS-PAGE with Coomassie staining of Tr/DCatch tetramers showing the tetramer mobilities in unboiled samples (left) and the subunit compositions in boiled samples (right). Mix refers to the input sample.

Figure 4

Validation of binding capacity of Tre3DTag1 and Tr3DCatch1. (A) SDS-PAGE with Coomassie staining of Tre3DTag1, showing the reactivity of DTag with free SpyCatcher and the ability of the Tre subunits to bind up to three biotinylated affibodies (bio-AffiIGF1R). (B) SDS-PAGE with Coomassie staining of Tr3DCatch1, showing the reactivity of DCatch with a SpyTag-bearing affibody (AffiHER2-SpyTag) and the ability of the Tr subunits to bind up to three biotinylated affibodies (bio-AffiIGF1R).

Figure 5

Generation of a SpyAvidin octamer and eicosamer. (A) Cartoon of tetramer, octamer, and eicosamer construction. (B) Reaction of Tre3DTag1 with Tr3DCatch1 to generate an octamer (lane 3), which can subsequently be purified via gel filtration (lane 4), analyzed by SDS-PAGE with Coomassie staining. (C) Mass spectrometry of the octamer. Peaks corresponding to the octamer are marked with circles, along with the charge state of the highest peak. (D) Reaction of DTag4 and Tr3DCatch1 to generate an eicosamer, which can subsequently be purified via gel filtration (far right lane), analyzed by SDS-PAGE with Coomassie staining. (E) Mass spectrometry of the eicosamer. Peaks corresponding to an eicosamer and a hexadecamer impurity are marked with circles, along with the charge state of the highest peak for each.

Figure 6

Eicosamers and octamers strongly stimulated T cell signaling. (A) Cartoon of how an eicosamer may bind multiple biotinylated peptide–MHC complexes. (B) T cell activation determined by flow cytometry of ERK phosphorylation, unstimulated or after stimulation with 25 nM (left panel) or 100 nM (right panel) cognate peptide–MHC incubated with the streptavidin tetramer (SAe4), traptavidin tetramer (Tre4), octamer, or eicosamer. Representative results from experiments run in duplicate on two separate days. The percentage of cells with staining above the threshold (dotted line) is marked. (C) T cell stimulation as in (B) but with control peptide–MHC.