Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Abstract

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

Fig. 1.

Spontaneous intermolecular amide bond formation by SpyTag. (A) Amide bond formation between Lys and Asp side chains. (B) Key residues for amide bond formation in CnaB2 shown in stick format, based on PDB 2X5P. (C) Cartoon of SpyTag construction. Streptococcus pyogenes (Spy) CnaB2 was dissected into a large N-terminal fragment (SpyCatcher, left) and a small C-terminal fragment (SpyTag, right). Reactive residues are highlighted in red. (D) SpyTag and SpyCatcher associated covalently. SpyTag-MBP and SpyCatcher were mixed each at 10 μM for 3 h and analyzed after boiling by SDS-PAGE with Coomassie staining, alongside unreactive controls, SpyCatcher E77Q (EQ) and D117A (DA) SpyTag-MBP.

Fig. 2.

Characterization of isopeptide bond formation. (A) Mass spectrometry of reconstitution between Cna peptide and SpyCatcher. The minor peak results from the His₆-tag on SpyCatcher leading to some gluconylation (see SI Appendix). (B) Time course of SpyTag-MBP:SpyCatcher covalent complex formation, with each partner at 10 μM at 25 °C, pH 7.0 determined by SDS-PAGE. (C) Rate constant for SpyTag reaction, calculated from triplicate measurements (each point shown) of SpyCatcher depletion under conditions as in (B). The equation for the trend-line and the correlation coefficient are shown.

Fig. 3.

Sensitivity of SpyTag reaction to conditions. (A) Temperature dependence of SpyTag-MBP reconstitution with SpyCatcher at 10 μM at pH 7.0 after 1 or 10 min. (B) pH-dependence of reaction as in (A) at 25 °C. (C) SpyTag-MBP and SpyCatcher were incubated each at 10 μM at 25 °C for 1 or 10 min at pH 7.0 in phosphate buffered saline (PBS), phosphate-citrate (PC), Tris, or Hepes buffers. (D) SpyTag-MBP and SpyCatcher were incubated each at 10 μM at 25 °C for 3 h at pH 7.0 in phosphate-citrate buffer containing no detergent (None), 1% Triton X-100 (Tx100), 1% Tween 20, or 0.5% Nonidet P-40 (NP40). All reactions were analyzed by SDS-PAGE and Coomassie staining. (All graphs mean of triplicate ± 1 s.d.; some error bars are too small to be visible.)

Fig. 4.

SpyCatcher reacted specifically. (A) SpyTag was reactive inside the E. coli cytosol. SpyTag-MBP and SpyCatcher were expressed in isolation or in the same cells. Cells were lysed and denatured by boiling with SDS buffer, before SDS-PAGE and Coomassie staining. SpyCatcher EQ was a negative control. To show that reconstitution happened within cells, lysates (lane 6) or cells (lane 7) expressing SpyCatcher alone (lane 1) or SpyTag-MBP alone (lane 3) were mixed. (B) SpyTag specificity inside the E. coli cytosol. Proteins were expressed as in (A) and the His₆-tags, present on SpyTag-MBP, SpyCatcher (EQ) and anything with which they have reacted, were pulled down with Ni-NTA, before SDS-PAGE and Coomassie staining. (C) Cartoon of targeting SpyTag to the mammalian cell surface, by genetic fusion to ICAM1 linked to GFP, with an HA tag after the signal sequence. (D) HeLa SpyTag-ICAM1-GFP were incubated with dye-labeled SpyCatcher for 15 min and analyzed by fluorescence microscopy. The GFP image (green, left) highlights cells expressing SpyTag-ICAM1-GFP, the 555 image (red, middle) shows the cells to which SpyCatcher bound, and the bright-field image (grayscale, right) shows all the cells. SpyCatcher EQ-555 (lower boxes) is a negative control. (Scale bar, 20 μm).

Fig. 5.

Dynamic force spectroscopy of the SpyTag:SpyCatcher interaction. (A) Cartoon of the constructs for testing the SpyTag:SpyCatcher interaction by AFM. (B) Representative force extension curve for Cys-I27₂-SpyCatcher forming a strong interaction with SpyTag-MBP-Cys, showing regions corresponding to PEG/agarose stretching, unfolding of two I27 domains (enlarged in the inset), and covalent bond breakage. (C) Histogram of different bond breakage forces for Cys-I27₂-SpyCatcher interactions with SpyTag-MBP-Cys. (D) Table of statistics of breakage forces. SpyTag:SpyCatcher was compared with the control SpyTag:SpyCatcher EQ. SpyTag was also tested against a cantilever only coated with PEG (SpyTag:PEG), or SpyTag was preblocked with free SpyCatcher before testing with a SpyCatcher-coated cantilever.

Fig. P1.

A domain of a protein involved in binding to human cells, from an invasive strain of Streptococcus pyogenes (Spy), was genetically dissected to generate a protein partner (SpyCatcher) and a peptide tag (SpyTag) (cartoon based on the crystal structure 2X5P). Upon mixing, SpyCatcher and SpyTag reacted rapidly and specifically to form a spontaneous amide bond, which was not reversed by boiling or mechanical stress. Fusion to SpyTag should provide a simple tool for irreversibly grasping proteins inside or outside cells.