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 , 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 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 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 ( 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 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. S7A). However, overnight incuba- of sodium dodecyl sulfate from the loading buffer for SDS-PAGE. tion with competing free peptide did not lead to a decrease in the SpyTag-MBP:SpyCatcher covalent complex and there was no ap- SpyTag was Reactive at N-Terminal, Internal, and C-Terminal Sites. pearance of the Cna peptide:SpyCatcher complex (SI Appendix, Antibodies often preferentially recognize peptide tags at specific Fig. S7B). Cna peptide gave complete conversion of unreacted termini (1), while split intein/sortase tags must be at particular SpyCatcher (SI Appendix, Fig. S7B, lane 8). Therefore, the Spy- sites in the protein (14, 15). To establish the generality of SpyTag Tag reaction did not reverse under these conditions. Note that function, we tested whether SpyTag could react at different loca- once the protein is unfolded the isopeptide bond between SpyTag tions in proteins. SpyTag-MBP has the SpyTag internally: SpyTag and SpyCatcher should be as chemically stable as a typical amide is preceded by a His6-tag and a thrombin cleavage site at the N bond, resisting prolonged boiling. terminus and is followed by MBP. N-SpyTag-MBP has SpyTag directly after the initiating formyl-: reaction of N-Spy- SpyTag Reacted Efficiently Inside Cells. Because SpyTag and Spy- Tag-MBP with SpyCatcher was still efficient (SI Appendix,Fig.S6A). Catcher are genetically encodable, we tested whether SpyTag-MBP We also generated a construct with two SpyTags: the first SpyTag and SpyCatcher could react together inside the cytosol of Escher- was between MBP and the three zinc fingers of Zif268, while the ichia coli. Cells were transformed with a plasmid encoding the second SpyTagwas right at the C terminus, following Zif268 (termed proteins individually or bicistronically, induced with IPTG, and

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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)at25°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 μMat25°Cfor3hat 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.)

Zakeri et al. PNAS Early Edition ∣ 3of8 Downloaded by guest on September 24, 2021 subsequently boiled with SDS loading buffer to prevent any ex vivo the cytosol. Using silver staining and anti-His immunoblotting reaction. Lysates were then analyzed by SDS-PAGE. A covalent as more sensitive probes for lower abundance interactions de- complex corresponding to the expected molecular weight of tected a small amount of a second protein pulled down in Spy- SpyCatcher:SpyTag-MBP could be clearly observed and little un- Catcher-expressing bacteria (SI Appendix, Fig. S8). reacted SpyTag-MBP remained (Fig. 4A). This covalent complex was not present when SpyTag-MBP was coexpressed with the con- SpyTag Reaction was Specific at the Mammalian Cell Surface. Stable trol SpyCatcher EQ (Fig. 4A). Mixing lysate or cells from bacteria and specific targeting of biophysical probes at the mammalian expressing SpyCatcher or SpyTag-MBP individually did not lead to cell surface is an important challenge in the study of receptor covalent complex formation (Fig. 4A), indicating that reaction was function (1, 15). SpyTag was targeted to the surface of HeLa cells not occurring ex vivo and consistent with SpyTagbeing able to react by genetic fusion to green fluorescent protein-labeled Intercellu- efficiently in the cytosolic environment. lar Adhesion Molecule-1 (ICAM1), to give SpyTag-ICAM1-GFP To address whether SpyCatcher had reacted with other cyto- (Fig. 4C). SpyTag-ICAM1-GFP trafficked efficiently to the plas- solic proteins, His6-tag containing proteins were pulled down ma membrane (Fig. 4D). Alexa Fluor 555-labeled SpyCatcher with Ni-NTA. SpyTag-MBP, SpyCatcher, and SpyCatcher EQ all added to the medium labeled GFP-positive cells, even those with contain an N-terminal His6-tag, and so we would expect to pull- low expression levels, but did not label neighboring nonexpres- down these proteins and any covalent complexes they formed. As expected SpyTag-MBP and SpyCatcher had efficiently reacted, sing cells (Fig. 4D), showing that there was little nonspecific depleting almost all SpyTag-MBP, but there was minimal pull- binding. In addition, cells expressing SpyTag-ICAM1-GFP were down of proteins of other molecular weights as assessed by Coo- not labeled by the negative control dye-labeled SpyCatcher EQ massie staining, indicating specificity of the SpyTag reaction in (Fig. 4D). SpyCatcher attachment to cells was resistant to acid

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).

4of8 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1115485109 Zakeri et al. Downloaded by guest on September 24, 2021 wash (SI Appendix, Fig. S9 A and B), suggesting that this cellular PNAS PLUS labeling was highly stable. The nature of the labeling was further explored by immuno- blotting against the HA tag on SpyTag-ICAM1-GFP, showing an adduct of SpyTag-ICAM1-GFP consistent with interaction with SpyCatcher and stable to boiling in SDS, indicating that the re- action was covalent (SI Appendix, Fig. S9C). To see if SpyCatcher had reacted with other cellular proteins, we blotted against the His6-tag on SpyCatcher. The anti-His blot showed a new band, corresponding to the molecular weight of SpyCatcher:SpyTag- ICAM1-GFP, while there was no detectable reaction on cells not expressing SpyTag-ICAM1-GFP (SI Appendix, Fig. S9C). These blots indicate that SpyCatcher showed high specificity to detect SpyTagamidst the diverse other surface proteins (17) and that the reaction was efficient at cellular expression levels.

SpyTag Formed Mechanically Stable Bonds with SpyCatcher. Because the environment of the SpyTag:SpyCatcher reactive residues low- ers the activation energy for amide bond formation, it is possible that the activation energy for amide bond hydrolysis could also be decreased. We showed that under mild conditions, in the pre- sence of excess competitor, there was no sign of breakage of the bond to SpyTag (SI Appendix,Fig.S7). However, it was possible that pulling on SpyTag would distort the energy profile and lead to efficient bond breakage (18), making the SpyTag system me- chanically labile. We assessed the mechanical stability of SpyTag at the single-molecule level, using dynamic force spectroscopy with

an atomic force microscope (AFM). We fused SpyCatcher to two BIOCHEMISTRY I27 domains, which provide a fingerprint from their characteristic unfolding force, to validate that one is observing specific cantilever bending from pulling on a single molecule (19) (Fig. 5A). For the interaction between SpyTag and SpyCatcher we ob- Fig. 5. Dynamic force spectroscopy of the SpyTag:SpyCatcher interaction. served traces with a breakage force >1 nN (Fig. 5 B and C), con- (A) Cartoon of the constructs for testing the SpyTag:SpyCatcher interaction sistent with mechanochemistry taking place as the cantilever by AFM. (B) Representative force extension curve for Cys-I272-SpyCatcher breaks a covalent bond when it retracts (18). The median breakage forming a strong interaction with SpyTag-MBP-Cys, showing regions corre- force for the covalent interaction was 1.9 nN, which is >20 times sponding to PEG/agarose stretching, unfolding of two I27 domains (enlarged stronger than streptavidin-biotin or antibody-antigen interactions in the inset), and covalent bond breakage. (C) Histogram of different bond I27 (20, 21). C-N and C-C bonds are not predicted to break until 5 nN breakage forces for Cys- 2-SpyCatcher interactions with SpyTag-MBP-Cys. (18), so it is likely that was not the isopeptide bond, nor another (D) Table of statistics of breakage forces. SpyTag:SpyCatcher was compared with the control SpyTag:SpyCatcher EQ. SpyTag was also tested against a can- bond in the protein which broke, but rather a weaker link in the tilever only coated with PEG (SpyTag:PEG), or SpyTag was preblocked with chain, such as a C-S bond linking the proteins to the cantilever/ free SpyCatcher before testing with a SpyCatcher-coated cantilever. bead. The nonreactive SpyCatcher EQ, after 1,940 tests with Spy- Tag, formed zero interactions above 500 pN (Fig. 5D, comparing and forms β-sheet hydrogen bonds. 100–200 pN is strong for a pro- SpyCatcher and SpyCatcher EQ: Fisher’s exact test P < 0.0001). tein-protein interaction (23,24) and, in concert with the isothermal As further negative controls, with no SpyCatcher on the cantilever titration calorimetry data (SI Appendix,Fig.S3), the AFM results or with SpyTagon the bead preblocked with free SpyCatcher, there suggest that the SpyTag:SpyCatcher interaction has unusual me- were also zero force traces above 500 pN (Fig. 5D), consistent with chanical strength even before the covalent bond forms. the specificity of the >500 pN interactions between SpyTag and SpyCatcher seen by AFM. Discussion The cantilever was lowered for 20 s, in which time we would Formation of an amide bond from reaction of a carboxylate with expect only a fraction of covalent bonds to form (Fig. 2B), so an amine is thermodynamically unfavorable under standard bio- that we could probe the early stages of the SpyTag:SpyCatcher chemical conditions (16); the equilibrium constant for two amino interaction. The unusually long contact times of this experiment acids reacting to form a dipeptide plus water is approximately made it challenging to avoid nonspecific interaction between the 10−3 (16). Hence proteases reach equilibrium with almost com- cantilever and the bead surface (22). Hence there was a low back- plete hydrolysis of their peptide substrates, while protein synthesis ground of tests where the polyethylene glycol (PEG) and agarose depends on ATP-dependent activation by amino-acyl linkers were extended for the negative controls, using no Spy- tRNA synthetases. Studies attempting to use proteases to catalyze Catcher or using preblocked SpyTag (Fig. 5D), giving force traces amide bond formation have established that factors moving the <500 pN. The frequency of PEG extensions for SpyCatcher EQ equilibrium towards bond formation are a low dielectric constant interacting with SpyTag was comparable to these two controls to facilitate deprotonation (removing the energetic cost from de- (Fig. 5D). Therefore there was no detectable interaction between hydration of the charges) and trapping the product, either by trans- SpyCatcher EQ and SpyTag in this experiment. However, the fre- fer to an organic phase, precipitation, or an exergonic binding quency giving force traces <500 pN for SpyCatcher interacting interaction (16). Therefore, for intermolecular amide bond forma- with SpyTag was significantly higher than for the other three con- tion by SpyTag, the orientation of the reactive groups, the hydro- ditions (chi-squared P < 0.0001) (Fig. 5D), supporting that the phobic environment, and proton-shuffling by Glu77 all accelerate 100–200 pN peak for SpyTag and SpyCatcher (Fig. 5C) was a spe- the reaction kinetics, but the environment created in the SpyTag: cific interaction. This peak was likely the force from a noncova- SpyCatcher complex is also likely to determine that the position of lent interaction between SpyTagand SpyCatcher, as SpyTagdocks equilibrium lies firmly on the side of bond formation; we found that

Zakeri et al. PNAS Early Edition ∣ 5of8 Downloaded by guest on September 24, 2021 SpyTag formed the covalent complex with high yield both in vitro nucleus, outside the cell, but also in the low pH endosomes and (Figs. 1 and 2) and inside E. coli (Fig. 4). lysosomes. This robustness of spontaneous isopeptide formation Isopeptide bond formation is a spontaneous posttranslational to various conditions may be because docking of the peptide and modification found in several extracellular proteins from Gram- protein partner could create a microenvironment buried from the positive bacteria, including human pathogens such as Staphylo- surface. Note that derivatizing SpyCatcher with an N-hydroxy coccus aureus, Corynebacterium diphtheriae, and Streptococcus succinimide (NHS) activated dye did not block reaction with pneumoniae (10). There has been little indication of how fast iso- SpyTag (Fig. 4D), suggesting a reduced accessibility of Lys31 to formation occurs, because the protein recovered exogenous labels, which will facilitate surface attachment of Spy- from recombinant expression had reacted to completion (12, 25). Catcher or conjugation with other biophysical probes. With this split system we provide the clearest evidence that reac- There are a number of ways to engineer peptides to react with tion can occur in minutes, consistent with the biological time scale small molecules, most commonly SNAP-tag (an engineered pro- for export of these proteins (13, 26). Regarding the evolution of tein tag for multiprotein labeling in living cells) and HaloTag (2, this posttranslational modification, our search for a fast reacting 4, 32, 33), but unlike SpyTag, these methods are not suitable for peptide tag showed that every change we tried to the residues in directing interactions with other proteins. Coiled coils can drive the C-terminal β-strand, even conservative changes pointing away specific protein association in living cells but have limited stability from the protein partner, greatly reduced the speed of reaction (34). Native chemical ligation is an elegant way to link proteins (SI Appendix, Fig. S2B), suggesting that the reactive (35, 36) but specificity and yield appear to be limited in cells (36). must be precisely aligned for reaction and that there is strong Sortase can be used to link together two proteins in vitro or to link evolutionary selection for the stability the bond provides to a peptide with a small molecule on the surface of living cells, but the protein. Because residues in the C-terminal β-strand could requires high [Ca2þ] (15), which is damaging in the cytosol or not be changed, we managed to increase the efficiency of reaction nucleus. Photoreactive amino acids are powerful for identifying by including an extra 5 C-terminal amino acids (SI Appendix, unknown binding partners (37,38,39) but (i) require UV which is Fig. S2), which were not seen in the crystal structure (11) but often damaging in living systems, (ii) do not react with a defined may have an interaction with the main domain, facilitating dock- partner, and (iii) typically give low yields (38). A protein contain- ing of the peptide with SpyCatcher. ing an artificial alkyne amino acid can react with another protein Our results also provide insight into the mechanical stability of containing an artificial azido amino acid (40) but the reaction spontaneous isopeptide bonds. The pilin Spy0128, has been ana- must be catalyzed by toxic CuI . Furthermore, the reaction would lyzed by dynamic force spectroscopy and shown to be resistant to not just be between the tagged proteins but also between the free >1;000 stretching because of its isopeptide bond, but forces pN amino acids. Split inteins can covalently splice proteins together (sufficient to break covalent bonds) were not explored (27). Also, and have the great advantage of leaving no trace after reaction in Spy0128 the reaction was between Lys and Asn (25, 28), rather (14, 41, 42), but must be located at defined termini and may un- than Lys and Asp for CnaB2 (16). Like Spy0128, in CnaB2 the dergo side reactions (43). Thus SpyTag has distinctive features in β isopeptide is between the N-terminal and C-terminal -strands, terms of flexibility of reaction conditions, specificity in cells, and so that, if the isopeptide does not break, then the force on the high yield of reaction, in comparison to existing chemical biology protein will not cause any extension of the protein chain. Inter- approaches for irreversible protein targeting. molecular reconstitution can be achieved with split Spy0128 but Limitations of SpyTag are that it is not traceless, in contrast to the partners react orders of magnitude slower, undergo side re- split intein-mediated ligation, and that the reaction rate is still actions, and the protein is undesirably large (29). The stability of far from the diffusion limit, although future work with phage or the SpyTag interaction before the isopeptide bond forms may re- β ribosome display (44) should be able to improve the on-rate. The late to the known mechanical strength of long parallel -strands, rapid on-rate makes the femtomolar affinity streptavidin-biotin where multiple hydrogen bonds have to break simultaneously interaction so useful for isolation of biotin-conjugates at trace (23). The resilience of SpyTag:SpyCatcher to pulling forces after concentrations (31, 45). However, the strongest noncovalent in- isopeptide bond formation suggests that the CnaB2 domain may teractions, such as streptavidin-biotin, can be broken in seconds be exposed to high force in bridging S. pyogenes to fibronectin by molecular motors (46, 47) or in milliseconds by shear forces during cell invasion (13). Precise attachment of proteins to AFM (48), so it has been very difficult to provide barriers or locks in tips, to understand protein folding and resistance to extension, is cellular systems using current tools. Molecular motors will not be usually achieved by introducing cysteines. However, carbon-sul- able to break the covalent bond formed by SpyTag, which should fur or sulfur-sulfur bonds break at lower forces than amide bonds be targetable to specific proteins, compartments or cell types, ex- (18) and introducing free cysteines often disrupts folding of pro- pressed from inducible promoters. Overall, SpyTag should enable teins containing ; instead fusing a protein of interest to new possibilities for cell biologists to probe the effect of force in SpyTag may prove a useful handle to allow AFM tips to pull on cells (49) and for biochemists to create new protein architec- specific proteins, even on the cell surface. tures (34). Peptide tags are a central tool in molecular biology (1). How- ever, the reversibility of peptide binding often compromises sen- Materials and Methods sitivity of immunoassays, stability of arrays/nano-assemblies, and In Vitro Reconstitution Reactions. Cloning, protein expression, statistical ana- purity of protein purification (1, 30, 31). SpyTag is a short peptide lysis, isothermal titration calorimetry, AFM, microscopy, in vivo reconstitu- with fundamentally different binding characteristics to peptide tion, mass spectrometry, and immunoblotting are described fully in the SI tags in current use, forming a covalent isopeptide bond to its Appendix. Briefly, SpyTag-MBP, SpyCatcher, and variants of these proteins protein partner. Reaction requires only mixing and is rapid, high were expressed in E. coli and purified using Ni-NTA resin. yielding, robust to experimental conditions, and shows good Amide bond formation between the protein and peptide binding part- specificity. The reactivity of SpyTag at 37 °C means that SpyTag ners was monitored by SDS-PAGE. To demonstrate covalent reconstitution μ can be used in live cell imaging of mammalian cells and also po- (Fig. 1D), proteins were mixed at 10 M in PBS pH 7.4 at 25 °C for 3 h. All tentially in transgenic mammalian systems. The reactivity at 4 °C, quantified reactions were performed in triplicate. To stop reactions, samples were heated in SDS loading buffer on a Bio-Rad C1000 thermal cycler at 95 °C combined with the resistance to commonly used detergents, for 7 min. SDS-PAGE was performed on 14% polyacrylamide gels, using an should allow the use of SpyTagfor pull-downs in cell lysate, where X-cel SureLock (Life Technologies) at 200 V for approximately 1 h. Gels were the low temperature minimizes . The range of pH stained with Instant Blue Coomassie stain (Triple Red Ltd.) and band inten- values over which SpyTag can react and the absence of Cys in sities were quantified using a Gel Doc XR imager and Image Lab 3.0 software SpyTag or SpyCatcher should facilitate its use in the cytosol, (Bio-Rad).

6of8 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1115485109 Zakeri et al. Downloaded by guest on September 24, 2021 Reactions for analyzing speed, pH-dependence, temperature-depen- PBS pH 7.4, or PBS pH 7.4 containing 1% Triton X-100, 1% Tween 20, or PNAS PLUS dence, and the reversibility of amide bond formation were performed by 0.5% Nonidet P-40. mixing 10 μM of each protein in 40 mM Na2HPO4 with 20 mM citric acid Concentration-dependence of amide bond formation was analyzed by pH 7.0 (phosphate-citrate) for the indicated time at 25 °C, or at the indicated incubating SpyCatcher and SpyTag-MBP both at 1, 5, or 10 μM in phosphate- temperature and pH. (PBS alone would not enable proper buffering over the citrate pH 7.0 at 25 °C for the indicated times. pH range explored.) For determining temperature dependence all reactions For analyzing the effect of reducing agent on amide bond formation, were incubated in a Bio-Rad C1000 thermal cycler at 4, 25, and 37 °C with a SpyCatcher and SpyTag-MBP were mixed at 10 μM in phosphate-citrate heated lid to prevent evaporation. pH 7.0 with or without 10 mM final concentration of freshly prepared dithio- To calculate the rate constant, SpyTag-MBP and SpyCatcher at 10 μM were threitol (DTT) for 10 min at 25 °C. mixed in triplicate in phosphate-citrate and incubated at 25 °C for 1, 3, or To determine how SpyTag reacted at terminal or internal locations, N-Spy- 5 min, in the linear part of the reaction. Samples were then heated to Tag-MBP or MBP-SpyTag-Zif-SpyTag was incubated with SpyCatcher or 95 °C for 7 min in SDS loading buffer and analyzed on 14% SDS-PAGE with Coomassie staining. Unreacted SpyCatcher concentration was quantified SpyCatcher EQ for 30 min at pH 7.0 in phosphate-citrate buffer at 25 °C and from band intensity as above. 1∕½unreacted Spy Catcher was plotted against analyzed by SDS-PAGE and Coomassie staining; proteins were present at time and a straight line, whose gradient corresponds to the second order rate 10 μM, except for those marked 3× which were present at 30 μM. constant, was fitted using the “LINEST” linear least squares curve-fitting Calculation of percent reconstitution was performed by dividing the in- routine in Excel. The units were converted from μM−1 min−1 to M−1 s−1. tensity of the band for the covalent complex by the intensity of all the bands The correlation coefficient and error bars to the gradient were calculated in the lane, then multiplying by 100. To analyze the speed of SpyCatcher:Spy- using the LINEST function in Excel. The reaction half-time was calculated from Tag-MBP covalent complex formation, the reduction in intensity of the band t1 ¼ 1∕k½SpyCatcher . 2 0 for SpyCatcher was monitored relative to a control not incubated with μ μ To test reversibility, 10 M SpyTag-MBP and 10 M SpyCatcher in phos- SpyTag-MBP. phate-citrate were incubated for 3 h at 25 °C to allow reaction to take place. μ 100 M Cna peptide (RSGAHIVMVDAGSR, made by solid-phase synthesis at Cell Culture and Labeling. HeLa cells were grown in DMEM with 10% Fetal Calf 98% purity by Insight Biotechnology Ltd.) was then added and incubated at Serum, 50 U∕mL penicillin and 50 μg∕mL streptomycin. HeLa cells were 25 °C for 16 h, before SDS-PAGE. In control experiments, 10 μM SpyCatcher or transfected with a plasmid encoding SpyTag-ICAM1-GFP using TurboFect SpyCatcher EQ was incubated with 100 μM Cna peptide for 3 h at 25 °C in the (Fermentas) following manufacturer’s instructions. After 48 h, transfectants same buffer. Also, 10 μM SpyCatcher or SpyCatcher EQ was incubated with were selected and maintained with 5 μg∕mL puromycin. HeLa cells stably ex- 10 μM SpyTag-MBP for 3 h at 25 °C in the same buffer. The survey of SpyCatcher reactivity against the series of peptide-MBP pressing SpyTag-ICAM1-GFP were washed with PBS containing 5 mM MgCl2 μ variants was performed by mixing each protein at 10 μM in PBS pH 7.4 (PBS-Mg) and incubated in PBS-Mg containing 1% BSA and 5 M SpyCatcher- and incubating at 25 °C for 30 min. Alexa Fluor 555 or 5 μM SpyCatcher EQ-Alexa Fluor 555 for 15 min at 25 °C. BIOCHEMISTRY For analyzing the effect of various buffers on the covalent reconstitution Cells were then washed twice in PBS-Mg at 4 °C and imaged live. between SpyCatcher and SpyTag-MBP, each protein was mixed at 10 μMin either phosphate buffered saline (PBS) pH 7.4, phosphate-citrate, 50 mM Tris ACKNOWLEDGMENTS. Funding was provided by the Clarendon Fund (B.Z., (tris-hydroxymethyl aminomethane) pH 7.0, or 50 mM Hepes [4-(2-hydro- J.O.F.), the National Institutes of Health (V.T.M., E.C.), the National Science xyethyl)-1-piperazine ethanesulfonate] pH 7.0 and incubated at 25 °C for 1 Foundation (V.T.M.), the James and Esther King Biomedical Research Program or 10 min. (V.T.M), the School of Biology of the University of St. Andrews (U.S-L.), Oxford For analyzing the effect of detergent on amide bond formation, Spy- University Department of Biochemistry (B.Z., E.C.C. and M.H.), St. Peter’s Col- Catcher and SpyTag-MBP were mixed at 10 μM for 3 h at 25 °C in either lege (B.Z.), New College (J.O.F.), and Worcester College (E.C.C. and M.H.).

1. Jarvik JW, Telmer CA (1998) Epitope tagging. Annu Rev Genet 32:601–618. 19. Marszalek PE, et al. (1999) Mechanical unfolding intermediates in titin modules. Nat- 2. Griffin BA, Adams SR, Tsien RY (1998) Specific covalent labeling of recombinant ure 402:100–103. protein molecules inside live cells. Science 281:269–272. 20. Moy VT, Florin EL, Gaub HE (1994) Intermolecular forces and energies between ligands 3. Allen KN, Imperiali B (2010) Lanthanide-tagged proteins–an illuminating partnership. and receptors. Science 266:257–259. Curr Opin Chem Biol 14:247–254. 21. Neuert G, Albrecht C, Pamir E, Gaub HE (2006) Dynamic force spectroscopy of the 4. Halo TL, Appelbaum J, Hobert EM, Balkin DM, Schepartz A (2009) Selective recognition digoxigenin-antibody complex. FEBS Lett 580:505–509. of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. 22. Celik E, Moy VT (2012) Nonspecific interactions in AFM force spectroscopy measure- J Am Chem Soc 131:438–439. ments. J Mol Recognit 25:53–56. 5. Huh WK, et al. (2003) Global analysis of protein localization in budding yeast. Nature 23. Crampton N, Brockwell DJ (2010) Unravelling the design principles for single protein – 425:686–691. mechanical strength. Curr Opin Struct Biol 20:508 517. 6. Stanfield RL, Wilson IA (1995) Protein-peptide interactions. Curr Opin Struct Biol 24. Huang J, Nagy SS, Koide A, Rock RS, Koide S (2009) A peptide tag system for facile – 5:103–113. purification and single-molecule immobilization. Biochemistry 48:11834 11836. 7. Korndorfer IP, Skerra A (2002) Improved affinity of engineered streptavidin for the 25. Kang HJ, Coulibaly F, Clow F, Proft T, Baker EN (2007) Stabilizing isopeptide bonds – Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the revealed in gram-positive bacterial pilus structure. Science 318:1625 1628. binding site. Protein Sci 11:883–893. 26. Ton-That H, Marraffini LA, Schneewind O (2004) Protein sorting to the cell wall envel- ope of Gram-positive bacteria. Biochim Biophys Acta 1694:269–278. 8. Houk KN, Leach AG, Kim SP, Zhang X (2003) Binding affinities of host-guest, protein- 27. Alegre-Cebollada J, Badilla CL, Fernandez JM (2010) Isopeptide bonds block the ligand, and protein-transition-state complexes. Angew Chem Int Ed Engl 42:4872–4897. mechanical extension of pili in pathogenic Streptococcus pyogenes. J Biol Chem 9. Horne WS, Gellman SH (2008) Foldamers with heterogeneous backbones. Acc Chem 285:11235–11242. Res 41:1399–1408. 28. Hu XQ, et al. (2011) Autocatalytic intramolecular isopeptide bond formation in gram- 10. Kang HJ, Baker EN (2010) Intramolecular isopeptide bonds: protein crosslinks built for positive bacterial pili: a QM/MM simulation. J Am Chem Soc 133:478–485. stress? Trends Biochem Sci 36:229–237. 29. Zakeri B, Howarth M (2010) Spontaneous intermolecular amide bond formation be- 11. Oke M, et al. (2010) The scottish structural proteomics facility: targets, methods and tween side chains for irreversible peptide targeting. J Am Chem Soc 132:4526–4527. outputs. Journal of Structural and Functional Genomics 11:167–180. 30. Huang J, Makabe K, Biancalana M, Koide A, Koide S (2009) Structural basis for 12. Hagan RM, et al. (2010) NMR spectroscopic and theoretical analysis of a spontaneously exquisite specificity of affinity clamps, synthetic binding proteins generated through – formed Lys-Asp isopeptide bond. Angew Chem Int Ed Engl 49:8421 8425. directed domain-interface evolution. J Mol Biol 392:1221–1231. 13. Amelung S, et al. (2011) The FbaB-type fibronectin-binding protein of Streptococcus 31. Rodriguez P, et al. (2006) Isolation of transcription factor complexes by in vivo biotinyla- – pyogenes promotes specific invasion into endothelial cells. Cell Microbiol 13:1200 1211. tion tagging and direct binding to streptavidin beads. Methods Mol Biol 338:305–323. 14. Zettler J, Schutz V, Mootz HD (2009) The naturally split Npu DnaE intein exhibits an 32. Nonaka H, Tsukiji S, Ojida A, Hamachi I (2007) Non-enzymatic covalent protein labeling – extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett 583:909 914. using a reactive tag. J Am Chem Soc 129:15777–15779. 15. Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL (2007) Sortagging: a 33. Uttamapinant C, et al. (2010) A fluorophore ligase for site-specific protein labeling – versatile method for protein labeling. Nat Chem Biol 3:707 708. inside living cells. Proc Natl Acad Sci USA 107:10914–10919. 16. Kullmann W (1985) Proteases as catalytic agents in peptide synthetic chemistry—shift- 34. Woolfson DN (2005) The design of coiled-coil structures and assemblies. Adv Protein ing the extent of peptide-bond synthesis from a quantite negligible to a quantite Chem 70:79–112. considerable. J Protein Chem 4:1–22. 35. Kent SB (2009) Total chemical synthesis of proteins. Chem Soc Rev 38:338–351. 17. McDonald CA, Yang JY, Marathe V, Yen TY, Macher BA (2009) Combining results from 36. Chattopadhaya S, Abu Bakar FB, Srinivasan R, Yao SQ (2008) In vivo imaging of a lectin affinity chromatography and glycocapture approaches substantially improves bacterial cell division protein using a protease-assisted small-molecule labeling the coverage of the glycoproteome. Mol Cell Proteomics 8:287–301. approach. Chembiochem 9:677–680. 18. Beyer MK, Clausen-Schaumann H (2005) Mechanochemistry: the mechanical activation 37. Chin JW, et al. (2002) Addition of p-azido-L-phenylalanine to the genetic code of of covalent bonds. Chem Rev 105:2921–2948. Escherichia coli. J Am Chem Soc 124:9026–9027.

Zakeri et al. PNAS Early Edition ∣ 7of8 Downloaded by guest on September 24, 2021 38. Lee HS, Dimla RD, Schultz PG (2009) Protein-DNA photo-crosslinking with a genetically 44. Griffiths AD, Duncan AR (1998) Strategies for selection of antibodies by phage display. encoded benzophenone-containing amino acid. Bioorg Med Chem Lett 19:5222–5224. Curr Opin Biotechnol 9:102–108. 39. Suchanek M, Radzikowska A, Thiele C (2005) Photo-leucine and photo-methionine 45. Laitinen OH, Hytonen VP, Nordlund HR, Kulomaa MS (2006) Genetically engineered allow identification of protein-protein interactions in living cells. Nat Methods avidins and streptavidins. Cell Mol Life Sci 63:2992–3017. 2:261–267. 46. Chivers CE, et al. (2010) A streptavidin variant with slower biotin dissociation and 40. Hancock SM, Uprety R, Deiters A, Chin JW (2010) Expanding the genetic code of yeast increased mechanostability. Nat Methods 7:391–393. for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/ tRNA pair. J Am Chem Soc 132:14819–14824. 47. Perumal SK, Raney KD, Benkovic SJ (2010) Analysis of the DNA translocation and 41. Shi J, Muir TW (2005) Development of a tandem protein trans-splicing system based on unwinding activities of T4 phage helicases. Methods 51:277–288. native and engineered split inteins. J Am Chem Soc 127:6198–6206. 48. Pierres A, Touchard D, Benoliel AM, Bongrand P (2002) Dissecting streptavidin-biotin 42. Tavassoli A, Benkovic SJ (2007) Split-intein mediated circular ligation used in the interaction with a laminar flow chamber. Biophys J 82:3214–3223. – synthesis of cyclic peptide libraries in E. coli. Nat Protoc 2:1126 1133. 49. Vogel V, Sheetz MP (2009) Cell fate regulation by coupling mechanical cycles to 43. Siebold C, Erni B (2002) Intein-mediated cyclization of a soluble and a membrane biochemical signaling pathways. Curr Opin Cell Biol 21:38–46. protein in vivo: function and stability. Biophys Chem 96:163–171.

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