Peptide tag forming a rapid covalent bond to a PNAS PLUS protein, through engineering a bacterial adhesin Bijan Zakeria,1, Jacob O. Fierera,1, Emrah Celikb, Emily C. Chittocka, Ulrich Schwarz-Linekc, Vincent T. Moyb, and Mark Howartha,2 aDepartment of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; bDepartment of Physiology and Biophysics, University of Miami, Miller School of Medicine, P.O. Box 016430, Miami, FL 33101-6430; and cBiomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, United Kingdom Edited by James A. Wells, University of California, San Francisco, CA, and approved January 17, 2012 (received for review September 21, 2011) Protein interactions with peptides generally have low thermo- a peptide representing the C-terminal β-strand containing the re- dynamic and mechanical stability. Streptococcus pyogenes fibro- active Asp and a protein partner derived from the rest of the pro- nectin-binding protein FbaB contains a domain with a spontaneous tein would allow the two partners to reconstitute and undergo isopeptide bond between Lys and Asp. By splitting this domain covalent reaction (Fig. 1C). This reaction occurred initially over and rational engineering of the fragments, we obtained a peptide hours for each species present at 10 μM, but by rational optimi- (SpyTag) which formed an amide bond to its protein partner (Spy- zation of the S. pyogenes (Spy) protein partner (termed SpyCatch- Catcher) in minutes. Reaction occurred in high yield simply upon er) (SI Appendix, Fig. S1) and the peptide tag (termed SpyTag)(SI mixing and amidst diverse conditions of pH, temperature, and Appendix, Fig. S2), reaction now occurred in minutes (vide infra). buffer. SpyTag could be fused at either terminus or internally and Mixing SpyTag fused to Maltose Binding Protein (SpyTag-MBP) reacted specifically at the mammalian cell surface. Peptide binding with SpyCatcher led to high yield of a product resistant to boiling was not reversed by boiling or competing peptide. Single-molecule in SDS (Fig. 1D). Mutation of the catalytic Glu77 in SpyCatcher dynamic force spectroscopy showed that SpyTag did not separate (EQ mutant) or the reactive Asp117 in SpyTag (DA mutant) abol- from SpyCatcher until the force exceeded 1 nN, where covalent ished covalent bond formation (Fig. 1D). Isothermal titration ca- bonds snap. The robust reaction conditions and irreversible linkage lorimetry showed that SpyTag DA-MBP and SpyCatcher formed BIOCHEMISTRY of SpyTag shed light on spontaneous isopeptide bond formation a noncovalent complex with a Kd of 0.2 μM(SI Appendix, Fig. S3), and should provide a targetable lock in cells and a stable module a high affinity for a peptide-protein interaction (6–8). for new protein architectures. Electrospray ionization mass spectrometry confirmed that reaction of the peptide with SpyCatcher led to covalent bond bacterial attachment ∣ chemical biology ∣ microbiology ∣ protein formation, with the combined mass 18 Da less than the sum of engineering ∣ single molecule biophysics the individual masses, from loss of water (Fig. 2A). We determined the efficiency of reaction between SpyTag- agging with peptides (e.g., HA, myc, FLAG, His6) is one of the MBP and SpyCatcher and saw high yielding and rapid reconstitu- Tmost common ways to detect, purify, or immobilize proteins tion, with more than 40% forming a covalent bond in the first (1–4). Peptides are very useful minimally disruptive probes (5) minute (Fig. 2B). The second-order rate constant was 1.4 × 103 Æ but they are also “slippery”- antibodies or other proteins typically 43 M−1 s−1 (SD, n ¼ 3) and the reaction half-time was 74 s bind peptides with low affinity and poor mechanical strength (Fig. 2C). Reaction was still rapid with each partner present at (6–9). We sought to form a rapid covalent bond to a peptide tag twofold or 10-fold lower concentration (SI Appendix, Fig. S4). without the use of chemical modification, artificial amino acids, or cysteines (disulfide bond formation is reversible and restricted SpyTag Reaction was Robust to Diverse Conditions. We characterized to particular cellular locations). how sensitive the reaction between SpyTag-MBP and SpyCatcher It has recently been found that Streptococcus pyogenes,likemany was to the sample conditions. The reaction proceeded similarly other Gram-positive bacteria, contains extracellular proteins stabi- at 25 °C as at 37 °C (Fig. 3A). Reaction was also efficient at 4 °C, lized by spontaneous intramolecular isopeptide bonds (10). Here although significantly slower than at 25 °C (for the 1 min time- we explored the second immunoglobulin-like collagen adhesin point: P < 0.0001, n ¼ 3) (Fig. 3A). domain (CnaB2) from the fibronectin binding protein FbaB, found We were also interested in the dependence on pH, to see in invasive strains of S. pyogenes (11,12) and essential for phago- whether SpyTag could be used in low pH compartments of the cytosis-like uptake of the bacteria by endothelial cells (13). CnaB2 cell, such as endosomes. Reaction was efficient at all pH values contains a single isopeptide bond conferring exceptional stability: tested from 5 to 8 (Fig. 3B). In fact, the reaction was slightly faster CnaB2 remains folded even at pH 2 or up to 100 °C (12). By split- at pH 5 and 6 than at pH 7 (comparing pH 6 with 7 for the 1 min ting CnaB2 into peptide and protein fragments, followed by time-point: P < 0.0001, n ¼ 3) (Fig. 3B). rational modification of the parts, we developed a peptide tag of 13 amino acids that rapidly formed a covalent bond with its protein partner (138 amino acids, 15 kDa) and characterized the condi- Author contributions: B.Z., J.O.F., E.C., U.S.-L., V.T.M., and M.H. designed research; B.Z., J.O.F., E.C., E.C.C., U.S.-L., and M.H. performed research; B.Z., J.O.F., E.C., U.S.-L., V.T.M., tions for reaction, cellular specificity of bond formation, and resi- and M.H. analyzed data; and M.H. wrote the paper. lience of the reacted product. Conflict of interest statement: M.H. and B.Z. are authors on a patent application regarding peptide targeting via spontaneous amide bond formation (United Kingdom Patent Results Application No. 1002362.0). Design of SpyTag for Rapid Covalent Bond Formation. Crystallogra- This article is a PNAS Direct Submission. phy and NMR have shown that CnaB2 forms a spontaneous Data deposition: The sequence reported in this paper has been deposited in the GenBank intramolecular isopeptide bond (11, 12); quantum mechanical/ database, www.ncbi.nlm.nih.gov/genbank (accession no. JQ478411). molecular mechanical calculations (12) indicate that the unpro- 1B.Z. and J.O.F. contributed equally to this work. 31 tonated amine of Lys nucleophilically attacks the carbonyl 2To whom correspondence should be addressed. E-mail: [email protected]. 117 77 carbon of Asp (Fig. 1A), catalyzed by the neighboring Glu This article contains supporting information online at www.pnas.org/lookup/suppl/ (Fig. 1B). We hypothesized that splitting the CnaB2 domain into doi:10.1073/pnas.1115485109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1115485109 PNAS Early Edition ∣ 1of8 Downloaded by guest on September 24, 2021 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 Spy- Catcher 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. Reaction efficiency was compared in the presence of a range of MBP-SpyTag-Zif-SpyTag). Both SpyTags were reactive, generating a buffers (PBS, phosphate-citrate, Hepes, Tris), but these had little doubly-branched protein with two SpyCatcher moieties covalently effect, indicating that the reaction was robust to buffer composi- attached (SI Appendix,Fig.S6B). tion (Fig. 3C). In particular there was no need for Ca2þ or Mg2þ, even though these divalent cations are important for the function SpyTag Reaction was Not Reversible over a Day. Spontaneous isopep- of many extracellular proteins, such as integrins or sortases. tide bond formation is most common between Lys and Asn (10), Because there are no cysteines in SpyTag or SpyCatcher, as ex- where there is loss of NH3, which may diffuse away and so help pected there was no effect of reducing agents on the reaction to promote irreversibility. For spontaneous isopeptide bond for- (SI Appendix, Fig. S5), so that reaction should be efficient in mation between Lys and Asp, there is loss of H2O but there is the cytosol/nucleus, secretory pathway, or outside the cell. approximately 55 MH2O present (at least at the surface of the We also tested the effect on the reaction of adding detergent, protein), which could make the SpyTag:SpyCatcher reaction re- to investigate if SpyTag could be used in cell lysates or in condi- versible (16) (SI Appendix, Fig. S7A). To test whether the SpyTag tions used to stabilize membrane proteins. There was no substan- reaction would reverse, we allowed SpyCatcher to react with tial effect on the reaction in the presence of high concentrations SpyTag-MBPand then, to see if there was any backward reaction, of nonionic detergents (Fig. 3D). We have not come across buffer we added a 20-fold excess of Cna peptide to capture any free conditions that impair SpyTag reaction, apart from the presence SpyCatcher (SI Appendix, Fig.