Probing the Recognition of Post-Translational Modifications by

Letters pubs.acs.org/acschemicalbiology

Probing the Recognition of Post-Translational Modifications by Combining Sortase-Mediated Ligation and Phage-Assisted Selection Till Teschke,† Bernhard Geltinger,† Alexander Dose,†,§ Christian Freund,‡,∥ and Dirk Schwarzer*,†,§ †

Departments of Protein Chemistry and ‡Protein Engineering, Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany § Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Str. 4, 72076 Tübingen, Germany ∥ Institute of Biochemistry and Chemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany S Supporting Information *

ABSTRACT: Reversible post-translational modifications (PTMs) are key regulators of protein function and modulate a multitude of protein−protein interactions in signal transduction networks. Here, we describe a strategy for determining the modification preferences of PTM-binding proteins with only minimal protein amounts that can be obtained by immunoprecipitation from mammalian cell lysates. This method bases on the combination of sortase-mediated ligation and phage-assisted selection strategies. This method can be used to analyze the type of modification that mediates the interaction as well as the influence of the amino acids flanking the modification sites. We have demonstrated the applicability of this method by probing the interaction of phosphorylated tyrosine and serine residues with their respective binding domains.

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Here, we report a simple strategy for determining the modification preferences of purified proteins or proteins immunoprecipitated from mammalian cell lysates by combining sortase-mediated ligation of peptides with a phage-assisted selection strategy. This method can be used to analyze the type of modification that mediates the interaction, e.g., phosphorylated serine, threonine, or tyrosine residues as well as the influence of the amino acids flanking the modification site. As mentioned above, the major challenge for such an approach is the limited amount of proteins obtained from cellular lysates. Therefore, an efficient amplification of the interaction readout signal is required. Phage-display is a very powerful technique for analyzing protein−protein interactions on a very small scale.5 Immobilized bait proteins can be probed with a library of randomized (poly-)peptide sequences displayed as fusion proteins on the surface of phages.6 Phages binding with sufficient affinity are enriched on the bait and are rescued by infecting E. coli. In the following, the phage genome encoding the interacting polypeptide is amplified by the proliferating infected E. coli cells. Despite its versatile applicability, phagedisplay per se is not suitable for probing modificationdependent protein−protein interactions because the displayed peptides and proteins are limited to the 20 amino acids of the ribosomal code.7,8 However, in recent chemical biology investigations, several chemoselective ligation strategies and bioconjugation techniques have been employed to link non-

eversible post-translational modifications (PTMs) mediate a multitude of protein−protein interactions that assemble protein complexes in almost all signal transduction networks of higher eukaryotes.1 Determining the modification preferences of such protein interfaces is crucial for a comprehensive understanding of cellular signaling processes. A common approach to analyze these interactions uses immobilized synthetic peptides containing the PTM of interest as baits for isolating binding proteins from cellular lysates.2 While these classic pull-down experiments are widely used, they bear the risk of a suppressed interaction between the PTM and a protein of interest (POI) due to the presence of other binders in the complex protein mixture. This risk increases when the POI is a protein of low abundance, which is the case for most signaling proteins and transcription factors. Ideally, the POI is purified from the lysate and characterized with different PTMcontaining binding partners in vitro. Most techniques employed for this purpose, including fluorescence polarization (FP) and isothermal titration calorimetry (ITC), rely on sufficient amounts of purified proteins that are commonly produced recombinantly in bacteria.3,4 However, many mammalian signaling proteins cannot be produced in bacteria as full-length constructs and with physiological activity. Alternative expression in the native host organism, mammalian cells, is generally low yielding, and the protein amounts required for the techniques mentioned above can hardly be obtained with standard procedures of tissue culture. To fill this technological gap, a general method needs to be established that is sensitive enough to readout the modification preferences of very low amounts of POIs. © 2013 American Chemical Society

Received: March 1, 2013 Accepted: May 30, 2013 Published: May 30, 2013 1692

dx.doi.org/10.1021/cb4001487 | ACS Chem. Biol. 2013, 8, 1692−1697

ACS Chemical Biology

Letters

Figure 1. Establishing sortase-mediated phage-display. (a) General scheme of sortase-mediated ligation reactions with peptides and proteins. (b) Strategy for determining the modification preferences of proteins immunoprecipitated from mammalian cells: At first, a set of G5-sortase presenting phages is generated. The displayed G5-sortase ligates any peptide containing the LPXTG sorting motif covalently to its own N-terminus and thereby the phage particle. In addition, the phages possess individual unique barcode DNA sequences that can be used to encode the ligated peptides. After ligation in separate vessels, the peptide-modified phages are pooled. The protein of interest is purified from mammalian cell lysates by immunoprecipitation and probed with the pooled phages. Phages displaying the preferred binding motif are enriched on the bound protein, and phages are subsequently rescued by infecting E. coli after elution. The infected cells amplify the barcode DNA, which after DNA sequencing yield the selected peptide.

Table 1. Peptides and Libraries barcode

a

peptide

sequencea

Fyn-lib-1

Fyn-lib-2

pep1 pep2 pep3 pep4 pep5 pep6 pep7 pep8 pep9 pep10 pep11

Ac-AcPQpYEEIPI-Ahx-LPKTGGRR-NH2 Ac-PQYEEIPI-Ahx-LPKTGGRR-NH2 Ac-PQpYREIPI-Ahx-LPKTGGRR-NH2 Ac-PQpYKAIPI-Ahx-LPKTGGRR-NH2 Ac-pY-Ahx-Ahx-LPKTGGRR-NH2 Ac-PQpSEEIPI-Ahx-LPKTGGRR-NH2 Ac-RRYRpSLPA-Ahx-LPKTGGRR-NH2 Ac-RRYRSLPA-Ahx-LPKTGGRR-NH2 Ac-RRYRQLPA-Ahx-LPKTGGRR-NH2 Ac-RRYRYLPA-Ahx-LPKTGGRR-NH2 Ac-RRYRpYLPA-Ahx-LPKTGGRR-NH2

B3 B2 B1 B4 B5 B6

B2 B6 B3 B1 B4 B5

14-3-3-lib-1

14-3-3-lib-2

FLAG-lib-1

FLAG-lib-2

B1 B4 B7 B2 B3

B4 B7 B1 B3 B2

B4 B2 B3 B1 B5

B1 B5 B4 B2 B3

Ahx (aminohexanoic acid) is used as a spacer.

phages display a sortase A mutant that is N-terminally extended by a penta-Gly tail (G5-sortase) facilitating highly efficient ligations of LPXTG-containing peptides to its own N-terminus and therefore the phage particle. Our strategy for probing modification-dependent protein−protein interactions is illustrated in Figure 1b: At first, a set of unique DNA sequences is inserted into the phagemid encoding the phages. These sequences serve as unique ‘barcodes’ and allow the identification of any peptide ligated to the displayed G5-sortase. Subsequently, the phages are ligated to synthetic peptides containing the desired modification (e.g., phosphorylation) imbedded in different recognition motifs. Afterward, the modified phages are pooled to form a library for probing the

natural moieties like synthetic peptides and small molecules to phage particles in order to encrypt them with the phage genomes.9−11 We have recently reported a phage-display system that should be ideally suited for probing modification-dependent protein−protein interactions.12 This system was initially established to evolve the substrate specificity of the bacterial transpeptidase sortase A.13 Sortases have gained considerable attention recently because they are able to ligate two unprotected peptides in a chemoselective manner.14−16 The only requirement is a so-called LPXTG sorting motif in one of the peptides and at least one Gly residue at the N-terminus of the other peptide (Figure 1a).17 In the established system, M13 1693



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modification preferences of a resin-bound protein immunoprecipitated from mammalian cell lysates. Phages displaying the preferred binding motif are selected from the librar,y and after infecting E. coli, the proliferating cells amplify this particular phage. The advantages of this system lie in the ability of the phages to perform a self-ligation reaction with any peptide containing the LPXTG motif. No activation or other chemical manipulation of the displayed peptides is required. Furthermore, we have previously shown that the self-ligation of G5sortase is preferred over a ligation in trans. This is an important feature ensuring stability of the library after pooling the phages because the transfer of modified peptide from one phage to another is unlikely, and indeed, we did not observe shuffling of ligated peptides (data not shown). In addition, the chemoselectivity of the reaction ensures ligation to the penta-Gly extension of the displayed G5-sortase, and in combination with a display rate adjusted to about one fusion protein per M13, phage avidity effects can be controlled. Finally, the barcoding DNA is shielded by the phage envelope protecting it from degradation and from interaction with the peptides used for probing the protein of interest. At first we tested the feasibility of this concept by probing the phosphorylation-dependent protein−protein interactions of the recombinant SH2 domain from the Fyn kinase.18 This domain binds phosphorylated tyrosine residues preferentially imbedded in a pYEEI motif.19 Two peptides derived from this consensus sequence were synthesized either in phosphorylated (pYEEI, pep1) or nonphosphorylated form (YEEI, pep2). The highly homologous Src SH2 domain binds tightly to the phosphorylated motif (Kd: 0.2 μM), while the nonphosphorylated sequence represents an inferior substrate (Kd: 2 mM).20 In addition, the peptides were C-terminally extended by the LPXTG sorting motif (see Table 1). A set of barcoded phages was generated by inserting unique 18−22 bp long sequences (B1−B7) into a noncoding region of the pCANTAB 5 E-SrtA phagemide (Table S1, Supporting Information). Infecting phagemid-transformed E. coli cells with VCSM13 helper phages generated the desired barcoded and potentially self-ligating phage particles. In contrast to conventional phage-display, this phage-selection strategy is limited to a single panning step because the modification state of the phage cannot be reestablished after rescue. Therefore, we analyzed the enrichment of phage-encoded phosphorylated versus nonphosphorylated peptides in a single pull-down experiment. Peptides pep1 and pep2 were added in large excess to the phages carrying barcodes B1 and B2, respectively, ensuring quantitative ligation. Phages were precipitated and washed extensively to remove excess peptides. In the following, 2 × 1012 infective particles of each phage were used in separate pull-down experiments with recombinant GST-Fyn-SH2 domain immobilized on GSHagarose (Figure 2a,b). We observed that about 200-fold more phages modified with the phosphorylated peptide (pep1, 2 × 107 colony forming units (cfu)) could be rescued after the pulldown reaction compared to phages displaying the nonphosphorylated substrate (pep2, 1 × 105 cfu). On the basis of this, we performed and analyzed a pull-down experiment with a mixture of both phages. Two × 1012 of each modified phage was mixed and used as input in the pull-down experiment. In total, 2 × 107 cfu were obtained, similar to the pull-down experiment with pep1-modified phage alone. In the following, 20 randomly selected colonies were analyzed by colony polymerase chain reaction (PCR) with oligonucleotides against either barcode B1 or B2. As illustrated in Figure 2c, only

Figure 2. Analysis of phospho-Tyr recognition by the Fyn-SH2 modified phages. (a) Phages recovered after pull-down experiment with phospho-Tyr (pep1) modified phages. (b) Rescued phages after pull-down experiment with nonphosphorylated (pep2) modified phages. (c) Analysis of a pull-down experiment with a mixture of phosphorylated and nonphosphorylated phages by colony PCR. Control without DNA (−), control with purified plasmid DNA of the respective barcode (+), and colony PCR of 20 randomly selected colonies.1−20 The top panel shows the analysis with an oligonucleotide against the barcode DNA encoding the phosphorylated pep1 and the bottom panel the analysis with an oligonucleotide against the barcode encoding the nonphosphorylated pep2.

barcode B1 could be amplified confirming that these colonies resulted from the rescue of pep1-modified phages. Encouraged by these results, we synthesized a small set of peptides to probe the selectivity profile of the Fyn-SH2 domain. In addition to the pY residue, the Fyn-SH2 domain requires two acidic residues for efficient substrate recognition. In pep3 and pep4, we mutated these residues altering the charge of the recognition site. In SH2 domains of the Src family, this leads to a decrease in binding affinity by one (Kd: 8 μM) and two (Kd: 80 μM) orders of magnitude, respectively.21 In pep5 and pep6, all other residues except pY were removed, and pY was replaced by pS, resulting in a further weakening of the binding affinity to the SH2 domain (Table 1).20 These peptides were separately ligated with phages carrying barcodes B1−B6 as described above and subsequently pooled to form the library Fyn-lib-1. After probing immobilized Fyn-SH2-domain with this library, colonies were further analyzed for their barcode content. Because of the increased number of barcodes, a more efficient readout strategy had to be established. To this end, we resorted to quantitative real-time PCR (qPCR), which is widely used in molecular biology for determining the amount of DNA or RNA in a given biological sample. All colonies obtained from the pull-down experiment were combined, and the phagemid DNA was extracted. In the following, we performed qPCR with oligonucleotides directed against each of the individual barcode sequences. As illustrated in Figure 3a, the barcode sequence of pep1 was amplified at earlier PCR cycles than the other barcodes, indicating a higher concentration of the B3 phagemid. A quantitative analysis of the qPCR confirmed that more than 90% of the input DNA belonged to the B3-coded phagemid. To ensure that variations in phage concentration and modification did not influence the readout, the same experiment was also performed with a second library (Fyn-lib-2) where pep1−pep6 were encoded by a different combination of 1694



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mammalian cells. HeLa cells were transiently transfected with an expression plasmid for FLAG-tagged 14-3-3ζ (Figure S2, Supporting Information). Cells of a single tissue culture dish (6 × 106) were transfected, and the FLAG-14-3-3ζ protein was immunoprecipitated from the lysate in anti-FLAG coated plates. After extensive washing the immunoprecipitated material was probed with FLAG-lib-1 and FLAG-lib-2, and the phagemids isolated from subsequently infected E. coli cells were analyzed by qPCR. As illustrated in Figures 3c and S1c, Supporting Information, the barcoded phagemids of pep7 still represented the dominant fraction with about 70% of the input DNA. The enrichment of these barcodes was lower than detected for the pull-downs with the recombinant proteins, which might reflect the lower amount of bait protein recovered from the HeLa cell lysate. However, the system still faithfully reported the binding preferences of the 14-3-3ζ protein under conditions usually used for coimmunoprecipitation experiments. In summary, we have established a strategy for probing the modification preferences of proteins immunoprecipitated from lysates of mammalian cells by combining sortase-mediated ligation with a phage-selection strategy. The displayed PTMcontaining peptides are accessible by standard methods of solid-phase peptide synthesis, and as yet, the method works for analyzing modification-dependent binding events with nanomolar affinities in a single panning step. Future optimization to allow the analysis of weaker interactions will take recent advantages in display technique into account. These techniques exploit avidity effects by defined display of dendrimers on phage surfaces.24−26 Furthermore, sortases were recently used to modify different surface proteins of the M13 phage, which might represent an additional method for the display of multiple copies of PTM-containing peptides.11 Importantly, the modified peptides used in this approach do not require activation or other chemical manipulation to facilitate ligation to the phage particles, and the features of this selection strategy ensures defined display of the respective modified peptides.12 We believe these features render this method broadly applicable for studying the interaction profiles of other modificationdependent protein−protein interactions in various biological systems.

Figure 3. Decoding the barcodes after the pull-down experiments with the phage-encoded Fyn and 14-3-3 library by qPCR. (a) Analysis of phagemids obtained from the pull-down experiment with recombinant GST-Fyn-SH2 and Fyn-lib-1. (b) Analysis of phagemids obtained from the pull-down experiment with recombinant GST-14-3-3ζ and 14-3-3lib-1. (c) Analysis of phagemids obtained from probing immunoprecipitated FLAG-14-3-3ζ with FLAG-lib-1 (Rn = normalized reporter fluorescence intensity).

B1−B6 barcoded phages. In this case, the B2-phagemid encoding pep1 represented over 90% of the extracted phagemids (Figure S1a, Supporting Information), which in summary confirmed the applicability of our approach. Next we used this system to analyze another modificationdependent protein−protein interaction: the recognition of phosphorylated serine residues by 14-3-3 proteins.22 The 14-33 proteins are universal adapters in eukaryotic proteomes that commonly bind their target proteins at sites of phosphorylated serine and threonine residues.23 A common binding motif is characterized by an RR[SFYW]XpS[WYFL]P sequence, which is recognized by 14-3-3 proteins with a Kd value of 0.1 μM.23 We synthesized a set of 5 peptides based on this motif introducing either pS, S, Q, Y, or pY at the modification site (pep7−pep11, Table 1). These peptides were ligated with barcoded phages as described above forming two libraries (143-3-lib-1 and 14-3-3-lib-2, Table 1) that were subsequently used to probe the binding preferences of recombinant GST-14-3-3ζ immobilized on GSH-agarose. After the pull-down and rescue of the phages, the phagemids of infected E. coli cells were analyzed by qPCR (Figures 3b and S1b, Supporting Information). In both cases, the barcoded phagemids encoding the strongest binder, pep7, represented over 95% of the input DNA. Finally, we analyzed if this system is capable of probing the modification preferences of proteins immunoprecipitated from

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METHODS

General Methods, Solid-Phase Peptide Synthesis, Protein Production, and Protein Immobilization. Detailed information is provided in the Supporting Information. Phage Production. The construction of the pCANTAB-G5-SrtA phagemid was described previously.11 The seven DNA barcodes listed in Table S1, Supporting Information, were generated from synthetic oligonucleotides and cloned into pCANTAB-G5-SrtA using the EcoO109I and AatII restriction sites. The identity of all constructs was confirmed by DNA sequencing. Seven clones, each containing a pCANTAB-G5-SrtA plasmid carrying one of the seven different DNA barcodes, were inoculated in 2xYT medium supplemented with ampicillin (100 μg mL−1), tetracycline (15 μg mL−1), and 2% w/v glucose. Cultures were grown at 30 °C and 180 rpm until the OD 600 reached 0.5. Cultures were then superinfected with 1010 to 1012 cfu of helper phage VCSM13 (Stratagene) for 30 min at 30 °C without agitation, centrifuged at 1500g and 25 °C, and the pellets were suspended in 2xYT with 100 μg mL−1 ampicillin, 35 μg mL−1 kanamycin, and 0.1 mM IPTG. Phage producing cultures were grown for 20 h at 30 °C and 180 rpm, and then centrifuged at 18 000g and 4 °C. Phages were precipitated from culture supernatants using 20% PEG/2.5 M NaCl, suspended in PBS supplemented with 25% glycerol, and finally shock frozen and stored 1695



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at −80 °C. After determining the titer of infective particles, phages were used for peptide ligation. Ligation of Peptides to G5-SrtA Presenting Phages. Unless stated otherwise, 5 × 1010 infective phage particles were used for ligation with 2.5 nanomoles of peptide for 16 h at 23 °C in a total volume of 50 μL. The 10× sortase ligation buffer contained 1.5 M NaCl, 50 mM CaCl2, and 500 mM Tris-HCl (pH 7.5). One hundred and twenty-five femtomoles of wild-type sortase A were added to ensure ligation. We ensured that the addition of excess wt sortase in trans did not decrease the overall ligation efficiency by direct hydrolysis of the ligation product or supply of free Gly nucleophile (Figure S3, Supporting Information). We performed 3 rounds of PBS washing and phage precipitation with 20% PEG/2.5 M NaCl to remove free peptide. Selection for Phage Displayed Peptides. Peptide presenting phages were pooled in libraries featuring five or six different peptides. Unless stated otherwise, approximately 5 × 1010 infective particles of each phage were used. Each target protein was tested separately with two libraries. Both libraries contained the same peptides and phages, but in different combinations. Libraries were incubated with the immobilized target protein for 1 h at 23 °C. Each well was washed 30 times with TBS-T and 30 times with TBS. Bound phages were eluted with 0.1 M glycine, pH 2.2, diluted, and used to infect a culture of E. coli XL1-Blue. Alternatively, bound phages were used to directly infect a culture of E. coli XL1-Blue that was added to each well for 30 min at 37 °C and 100 rpm. Cultures were plated on LB agar supplemented with ampicillin (100 μg mL−1) and 2% w/v glucose, and grown for 16 h at 37 °C. Directly infected cultures were diluted before plating. Phagemid DNA was extracted from all resulting colonies and used for real-time PCR. Quantitative Real-Time PCR. Real-time PCR was performed using SYBR Green I dye, the phagemid mixture extracted from the bacterial colonies, and primers targeting the DNA barcodes. Each barcode was tested in a separate reaction. Phagemid DNA of one barcode was quantified with PicoGreen dsDNA quantitation reagent (Life Technologies) and used as a standard. Sample reactions were prepared in duplicate, standard reactions in triplicate. The original amount of a specific phagemid in the extracted phagemid mixture was calculated from the cycle threshold value using the linear equation of the standard curve.

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ASSOCIATED CONTENT

S Supporting Information *

Additional methods and supporting figures and tables. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(D.S.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank K. Piotukh for support with phage-display techniques and L. Schmohl for assistance with ligation experiments. We further thank the Emmy−Noether Program of the Deutsche Forschungsgemeinschaft (DFG) (SCHW 1163/3-1) and the DFG SFB958 program (TP7) for financial support.

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REFERENCES

(1) Walsh, C. T., Garneau-Tsodikova, S., and Gatto, G. J., Jr. (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem., Int. Ed. 44, 7342−7372. (2) Elion, E. A. (2006) Detection of protein−protein interactions by coprecipitation. Curr. Protoc. Mol. Biol. 20, 20.5. 1696



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(24) Helms, B. A., Reulen, S. W., Nijhuis, S., de Graaf-Heuvelmans, P. T., Merkx, M., and Meijer, E. W. (2009) High-affinity peptide-based collagen targeting using synthetic phage mimics: from phage display to dendrimer display. J. Am. Chem. Soc. 131, 11683−11685. (25) Lempens, E. H., Merkx, M., Tirrell, M., and Meijer, E. W. (2011) Dendrimer display of tumor-homing peptides. Bioconjugate Chem. 22, 397−405. (26) Bastings, M. M., Helms, B. A., van Baal, I., Hackeng, T. M., Merkx, M., and Meijer, E. W. (2011) From phage display to dendrimer display: insights into multivalent binding. J. Am. Chem. Soc. 133, 6636− 6641.

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Probing the Recognition of Post-Translational Modifications by

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