Versatile Site-Selective Protein Reaction Guided by WW Domain

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Versatile Site-Selective Protein Reaction Guided by WW Domain− Peptide Motif Interaction Miao Liu,† Zeyang Ji,‡ Mingjie Zhang,‡ and Jiang Xia*,† †

Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China

‡

S Supporting Information *

ABSTRACT: A short, flexible, and unstructured peptide tag that has versatile and facile use in protein labeling applications is highly desirable. Here, we report an 11-residue peptide tag with an internal cysteine (a W-tag, derived from a Comm PY peptide motif that is known to bind with Nedd4 WW3* domain) that can be installed at different regions of the target protein without compromising its covalent reactivity with the reactive label (a 35-residue synthetic Nedd4 WW3* domain derivative). This versatility is explained by the unique structural features of the reaction. NMR analysis reveals that both the W-tag peptide and reactive Nedd4 WW3* protein are unstructured before they encounter each other. The binding interaction of the two induces noticeable structural changes and promotes global folding. Consequently, the reactive cysteine residue at W-tag and the electrophilic chloroacetyl group at Nedd4 WW3* domain are positioned to be in close proximity, inducing an intermolecular covalent cross-linking. The covalent linkage in turn stabilizes the folding of the protein complex. This unique multistep mechanism renders this labeling reaction amenable to different sites of the proteins of interest: installation of the tag at N- and C-termini, in the flexible linker region, in the loop region, and the extracellular terminus of target proteins exhibited comparable reactivity. This work therefore represents the first proximity-induced cysteine reaction based on the unique binding features of WW domains that demonstrates unprecedented versatility.

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peptide AP (the substrate of the biotin ligase BirA),17 a 13amino-acid sequence LAP (the substrate of a mutant lipoic acid ligase),18 a 7-residue Q-tag (the substrate of a transglutaminase),19 the small sortagging motif LPXTG (the substrate of a sortase),20 a LCTPSR formyl glycine tag (the substrate of a formyl glycine generating enzyme),21 and the peptides A1 and S6 (the substrates of different PPTases).22 Although the complexity of the tags is significantly reduced, their successful labeling requires the assistance of external enzymes. Without an enzyme to safeguard specificity and reactivity, site specific labeling of a short peptide tag requires the formation of a unique structural feature by the sequence of the peptide tag to vest one of its residues with outstanding reactivity. For example, a serendipitous discovery by Pentelute and co-workers identifies a π-clamp with an exceptionally small motif of only four residues which can specifically react with perfluoroaromatic reagents.23 Alternatively, through pretargeting, a mild reactive group can be positioned to the vicinity of a

INTRODUCTION It is challenging to establish a residue-accurate chemical reaction with only one specific amino acid residue among the same type of residues or the ones with similar reactivity. Active site residues of enzymes are bestowed special reactivity as the backbone of the protein framework forms a microenvironment around the active site residue that alters the chemical property of the amino acid. This feat has inspired chemists to design a covalent labeling strategy using the enzyme itself as the tag, with the corresponding labels being derivatives of suicide inhibitors, cofactors, or substrate analogs.1−7 Examples include activity-based protein probes,8,9 CoA-affinity-based kinase tags,10 the enzyme−suicide substrate-based SNAP/CLIPtags,11,12 the Halo-tag,13 the lactam-based β-lactamase-tag,14 and the small molecule inhibitor-based TMP tag. 15,16 Notwithstanding their outstanding specificity, the enzyme tags are normally cumbersome, and sometimes affect the function of the target protein. Alternatively, short peptide sequences from natural substrates of ligases or transferases can be converted to covalent tags, and the enzyme as a facilitating reagent will bring in another component, a labeling reagent, to covalently tether with the tag. Examples in this category include a 15-amino-acid receptor © XXXX American Chemical Society

Received: June 13, 2017 Revised: July 11, 2017 Published: July 12, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00334 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry natural amino acid to induce a site-specific reaction, a strategy called “proximity-induced reaction”.24−36 The advantage of proximity-induced reaction lies at its modularity and adaptability to a set of orthogonal tags. However, in most cases the tag is fused to one terminus of the protein, and intermolecular labeling at linker or loop region has not been demonstrated. We previously demonstrated proximity-induced cysteine conjugation reactions guided by coiled coil interaction,28 SH3-peptide,29,30 or PDZ-peptide binding interactions.29,31 However, all these binding pairs have limitations in the applications: the 21-residue CCE peptide tag still has a tendency to form bundles with other helices in the target protein, therefore potentially affecting its folding; SH3 and PDZ domain as tags are more than 100 residues, still very cumbersome, and therefore not amenable for labeling at linker or loop region. Here we turn to WW domain, one of the smallest protein−protein interaction domains (with only 35 residues), which binds to a short peptide sequence of ∼10 amino acids.37 Considering the sizes of the both binding partners, it is one of the smallest protein−peptide binding models. The small tag (∼10 residues) is expected to pose minimal structural disturbance to the target protein, and the label (∼35 residue WW domain) is accessible through synthesis and amenable for derivatization. Synthetic WW domains have been shown to retain their ligand binding properties,37,38 even when extensive mutagenesis was included.39 So, we design an 11-residue tag with an internal cysteine residue derived from a WW ligand Comm PY motif, to covalently react with a synthetic dNedd4 WW3* domain containing a chloroacetyl group. We then provide structural information on the labeling reaction, and prove that the tag can be inserted at termini, linker, and loop region with effective labeling.

Figure 1. WW domain guided covalent cysteine conjugation. (A) Structure of dNedd4 WW3* domain and Comm PY motif binding interaction (PDB ID 2EZ5). (B) Design of proximity-induced cysteine conjugation. (C) Reaction between synthetic WW domains and cysteine containing peptides. WWWT or WWX proteins were mixed with 4-fold excess of peptides at 37 °C overnight. The reaction mixtures were then resolved by denaturing SDS-PAGE and stained by Coomassie blue dye. Molecular weight shift is only observed in the mixture of WWX and pNC, indicating a covalent conjugation.

Table 1. Sequence of Synthetic Wild Type and Mutant Peptides, and WW Domainsa

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RESULTS AND DISCUSSION Design of the W-tag. We chose dNedd4 WW3* domain and the Comm PY peptide motif as the design template because of the extensive revelation of their complex structure (Figure 1A, PDB ID: 2EZ5).40 Solution NMR structure of the complex indicates that the glycine residue at N-terminus of PY motif (G228) and the asparagine residue at the β1/β2 loop (N542) of dNedd4 WW3* domain are in close proximity, with a distance of 3−4 Å between the α carbons. Notably, the PY motif peptide adopts a relaxed conformation, indicating that it is structurally favorable as a potential protein tag. Replacing G228 to Cys converts the PY motif peptide (pWT, with a sequence of TGLPSYDEALH) to a peptide tag with a nucleophilic group cysteine (pNC, TCLPSYDEALH). Another peptide pCC, TGLPSYDECLH, with a cysteine installed at a distant C terminal position was included as a control. On the other side, we replaced N542 of the dNedd4 WW3* domain with an α-chloroacetyl-containing unnatural amino acid (2S)-2amino-3-[(2-chloroacetyl)amino]propanoic acid (X) and converted the wild type dNedd4 WW3* domain (WWWT) to a reactive WW domain as a label (WWX, Table 1). The unique extensive interactions found in the complex between PY motif and dNedd4 WW3* are expected to maintain the binding affinity and specificity of the mutants. All five peptides including the WW proteins were synthesized by Fmoc solid-phase peptide synthesis, purified, and confirmed by mass spectrometry. We then measured their spontaneous reaction at 37 °C in PBS (pH 7.4). Only the incubation of WWX and pNC yielded a higher molecular weight band on SDSPAGE (Figure 1C, lane 3), indicating that the cysteine-

a

X = (2S)-2-amino-3-[(2-chloroacetyl)amino]propanoic acid.

chloroacetyl reaction of WWx with pNC follows a proximitydriven fashion; when the cysteine is not in the proximity of the chloroacetyl group in the case of WWx with pCC, no reaction could be observed. The covalent reaction was further confirmed by HPLC and ESI-MS analysis (Figures S1 and S2 in Supporting Information). We have demonstrated that proximity-induced cysteine-chloroacetyl reactions retain their specificity in 1−10 mM concentration of glutathione and inside the cytosol of mammalian cells.28,31,32 Structural Rearrangement during Cysteine Conjugation. To investigate how the reaction proceeds, we performed solution NMR analysis using the synthetic WW domains (WWWT and WWX) and PY peptides (pWT and pNC). Figure 2A shows the 1D 1H spectra of synthetic WWWT domain alone and in the presence of 0.5, 1, and 2.5 equiv of pWT peptide, and synthetic WWX domain alone and in the presence of 0.5, 1, and 4 equiv of pNC peptide. Several conclusions could be drawn from spectrum analysis: 1. Both PY peptides (pWT and pNC) are highly flexible and unstructured, evidenced by the extremely sharp peaks at around 7.9, 7.1, 7.0, and 6.75 ppm (red lines in Figure 2A) and peak intensity increase dramatically upon adding peptide. 2. The apo forms of both WW domains mainly exist as unfolded random coils, as indicated by overall narrowly dispersed peaks (around 6.7 to 8.0 ppm). 3. Peptide binding (in both pNC to WWX and pWT to WWWT cases) induces protein folding, shown by better peak dispersion pattern (from 6.2 to B

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Figure 2. Structural analysis of the WW-guided conjugation by NMR. (A) 1D 1H NMR spectra showing the titration of peptides into WW domains. The red lines at around 7.9, 7.1, 7.0, and 6.75 ppm indicate the signal from the PY peptides (pWT and pNC). The blue dashed lines at −0.843, 6.4, and 6.65 ppm indicate that the covalent cross-linking of the peptide with the protein promotes the folding of the complexes. (B) 2D 1H−1H NOESY spectra show −0.843 ppm peak only showed up in the pNC and WWX pair. (C) 2D 1H−1H NOESY spectra of the region corresponding to β-strand HAs (5.1 to 5.6 ppm) show more NOEs in covalently cross-linked pNC and WWX pair (shown in red), indicating better folding than the wild type pWT and WWWT pair (shown in green).

Figure 3. Covalent protein labeling at termini. (A) Reaction of N-terminal W-tag with WWX, with a CCE-1 tag giving no reaction, shown in SDSPAGE (the small band at 25 kDa is likely a truncated version of W-tagN-EGFP that co-purified with W-tagN-EGFP). (B) Both N-terminal and Cterminal W-tag reacted with reactive WWX domain, shown as a molecular shift in SDS-PAGE. [WW domain] = 40 μM, [Protein] = 10 μM. Reactions were in PBS buffer pH 7.4 at RT overnight.

8.4 ppm). 4. The covalent linkage assists the folding of WWX domain in its complex. This is shown by a unique chemical shift peak at −0.843 ppm and 6.4 and 6.65 ppm only in WWX−pNC system (dashed blue lines, which is indicative of folded residues). Such chemical shifts were not observed in wild type WW domains and the noncovalent complex. We next carried out 2D-NOESY spectra to further investigate the folding state of apo forms, WT complex, and covalently linked complex. First, consistent with the 1D spectra, it is confirmed that the −0.843 ppm peak (peaks with such unique chemical shift usually indicate that this residue is located in a well-folded region) only shows up in the covalent complex, but not in the noncovalent one (Figure 2B). Second, since βstrands usually show characteristic downfield chemical shifts of HA and WW domain is formed by 3 antiparallel β-strands, we then focused on the 1H range only favored by β-strand HAs (Figure 2C). In the strips of 1H 5.1 to 5.6 ppm, the WWX−pNC covalent complex shows more NOE signals than the WT

complex, and no NOE signals were observed for the apo-form. This means the following: (1) Both WW domains are mainly unfolded in the apo form. (2) Peptide binding facilitates the formation of β-strands, but the complexes are still not fully folded. (3) Covalent linkage in the WWX−pNC complex stabilizes the structure and promotes the folding. Taken together, the NMR analysis demonstrated that the conjugation between pNC and WWX followed a binding-inducing-folding and reaction further stabilizing folding mechanism. Covalent Protein Labeling at Termini. We then renamed pNC as W-tag, and examined the covalent labeling of W-tag fusion proteins, an enhanced green fluorescent protein (EGFP) as an example. We previously reported coiled-coil guided cysteine conjugation on a single cysteine residue at a CCE peptide fused to the N-terminus of EGFP. Here we fused W-tag to the N-terminus of EGFP and examined the specificity of the covalent labeling reaction by incubating the fusion proteins with synthetic WWX peptide. Only W-tagN-EGFP was found to C

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Figure 4. Covalent protein labeling at linker region and at a loop region. (A) Reaction of an internal W-tag between GST and HP1β with WWX label, shown as molecular weight shift in SDS-PAGE. (B) Reaction of an internal W-tag between MBP and HP1β with WWX. (C) Reaction kinetics of the internal W-tag in GST-W-tagINT-HP1β, quantified from SDS-PAGE. (D) Reaction kinetics of the internal W-tag in MBP-W-tagINT-HP1β, quantified from SDS-PAGE. (E) Reaction of a W-tag inserted into a loop region of EGFP with WWX label, as compared with that of a W-tag at Nterminus of EGFP. [WW domain] = 40 μM, [Protein] = 10 μM. Reactions were in PBS buffer pH 7.4 at RT overnight.

react with WWX (Figure 3A). As both EGFP and CCE-1 tag have other solvent-exposed cysteines on their surface, this indicates that only the cysteine of W-tag is reactive. This then proves that the reaction follows a proximity-induced mechanism, and the CCE1 and W-tag are two orthogonal covalent reactions. We then examined whether W-tag can be positioned to either terminus of the protein. For this purpose, we chose to tag the chromo domain of HP1β, which can bind with methylated lysine containing a protein sequence.41 W-tag was fused at either the N-terminus or C-terminus of HP1β to give W-tagNHP1β and HP1β-W-tagC. Both proteins were found to react with WWX with almost the same reactivity (giving a yield of 62% for W-tagN-HP1β and 52% for HP1β-W-tagC, Figure 3B). Covalent Protein Labeling at Internal Linker and Loop. Affinity tags are commonly attached to the termini, including His-tag, GST tag, HA tag, and MBP tag,42 as covalent

protein labeling reactions at internal regions are rare. Here we constructed two model systems that allow the internal linker regions to be covalently labeled. Two fusion proteins GST-WtagINT-HP1β and MBP-W-tagINT-HP1β with W-tag installed between the affinity tag protein and HP1β CD domain were used as model proteins. The 11-residue W-tag is very small compared to the overall 40 kDa and 60 kDa fusion proteins, but the reactivity of the internally positioned W-tag was similarly maintained in both cases (Figure 4A−D). WWX did not show noticeable reactivity with GST alone at WWX:protein ratio of 4:1 or lower at 37 °C (Figure S3 in the Supporting Information). Next we examined whether the 11-residue W-tag can be positioned in the loop region of a protein. We inserted WW tag at Y145 of EGFP, which was located at the β6/β7 loop.43 Although W-tag insertion changed the absorption spectra of EGFP, the fluorescence of EGFP was maintained (Figure S4 in D

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Figure 5. Fluorescent labeling of a W-tag fused EGFR on cell surface. Confocal fluorescent images of CHO cells expressing HA-W-tagEXTRA-EGFR and treated with 1 μM TAMRA-WWX label (overnight) and FITC-anti-HA antibody (2 h) sequentially. Scale bar = 10 μm.

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the Supporting Information). The WWX label readily reacts with the W-tag inserted in the loop region, and the protein attachment did not significantly alter the fluorescent property of the EGFP mutant (Figure 4E and Figure S4 in the Supporting Information), although the protein complex shows band splitting on SDS-PAGE, possibly due to the presence of multiple conformations after the WW protein is fused with EGFP. Covalent Protein Labeling on Cell Surface. Last, we examined if W-tag can be used for covalent protein labeling on the surface of cells. WW tag was inserted into the N-terminus of the extracellular portion of a modified epidermal growth factor receptor (EGFR) to generate a fusion receptor HA-WtagEXTRA-EGFR. A pDisplay vector was used to express the recombinant receptor in the plasma membrane of Chinese Hamster Ovary (CHO) cells. W-tag will be sandwiched between an N-terminal HA tag and the transmembrane domain. W-tag can be labeled by a red fluorescent synthetic WWX protein and with a green fluorescent anti-HA antibody simultaneously. After peptide and antibody labeling, CHO cells expressing HA-W-tagEXTRA-EGFR exhibited both green and red fluorescence on the plasma membrane (Figure 5). The TAMRA-WWX protein only labels the cells that express HAW-tagEXTRA-EGFR, indicating that the labeling reaction is specific toward the W-tag. Some red signal was found in the cytosol, likely caused by receptor-mediated internalization during peptide labeling on the live cells.28,44 Notably, peptide labeling was completed in cell culture medium and persisted after extensive washing steps.

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EXPERIMENTAL PROCEDURES WW Domain Guided Conjugated Reaction. The synthetic peptides were dissolved in phosphate buffered saline (PBS). The concentration was determined by UV−vis spectrometry using the absorption at 280 nm, and the absorption coefficient was calculated by ExPASy ProtParam tool (http://web.expasy.org/protparam/). 30 μM WW domain was mixed with 120 μM peptide ligand in PBS containing 1 mM tris(2-carboxyethyl)phosphine (TCEP) and incubated at 37 °C overnight. The reaction solutions were denatured at 95 °C for 10 min in the presence of loading dye, and resolved by tricine-SDS-PAGE. The gel was stained by Coomassie Blue. NMR Analysis of WW Domain. NMR samples were dissolved in PBS in D2O. The concentrations of NMR samples were 0.1 mM for 1D spectra, 0.5 mM for WWWT NOESY spectra, and 0.3 mM for WWX NOESY spectra. NMR spectra were acquired on Varian Inova 750 or 800 MHz spectrometers at 30 °C. Mixing times of 2D NOESY experiments were 200 ms. Spectra were processed using NMRPipe software46 and analyzed using NMRPipe and Sparky.47 Covalent Protein Labeling and Kinetics Measurement. All proteins were dissolved in PBS and the concentration was determined by UV absorption at 280 nm. 10 μM protein was mixed with 40 μM WW domain in PBS containing 1 mM TCEP and incubated at RT overnight. Reactions were resolved by glycine-SDS-PAGE stained by Coomassie Blue. For kinetics measurements, reaction solutions incubated at RT for different time periods were thermally denatured in the presence of loading dye, and resolved by SDS-PAGE. The gels stained by Coomassie Blue were imaged by Odyssey imaging system (LICOR Inc., USA). The protein bands were quantified by gray scale using Image Studio software. The reaction progression curves of the normalized yields were fit with an exponential increase equation (MnMolecular1, y = A1 − A2 × exp(−kappx), where A1 is the normalized maximal conjugation yield) using Origin software, and apparent reaction rates (kapp) were derived from the fitting. Cell Culture, Transfection, and Labeling. Chinese Hamster Ovary (CHO) cells were grown in DMEM/F12 medium (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12, Life Technology, USA) supplied with 10% fetal bovine serum (FBS, Life Technology, USA) in a 10 cm culture dish (Corning, USA) and maintained at 37 °C in a humidified incubator supplied with 5% CO2. For cell labeling, 1.0 × 105 cells were seeded in a 35 mm confocal dish (ibidi, Germany) 1 day prior to the transfection. Cells were transfected with 1 μg/mL DNA using FuGENE HD Transfection Reagent (Promega Corporation, USA) according to the manufacturer’s instructions. After 36 h, the cells were pretreated with PBS containing 0.5 mM TCEP for 10 min, and then incubated with fluorescent peptide probes in DMEN/F12 with 10% FBS at 37 °C overnight. The cells were then washed,

CONCLUSIONS

A protein tag that can achieve efficient labeling at N- and Ctermini, interdomain linker region, and intraprotein loop region is difficult to achieve.42,45 In particular, to the best of our knowledge, none of the proximity-induced reactions has demonstrated equivalent labeling efficiency at all these locations. Uniquely, the WW domain interactome harbors a great variety of binding partners with exceptionally small sizes (35 residues for WW domains and 10 residues for the binding peptides). We also found that the binding interaction of synthetic Nedd4 WW3* domain and the Comm PY peptide motif adopts a binding-induced structural transition from unstructured conformations to a well-folded complex structure. In this report, we translated this structural feature to the development of the versatality of covalent protein reactions: the flexible cysteine-containing W-tag can be incorporated into termini, linker, loop, and extracellular portion; the fusion proteins exhibit similar reactivity toward the synthetic chloroacetyl containing WWX label, through a binding-inducing folding and proximity-induced reaction mechanism. Our work represents the first rationally designed cysteine reaction with a wide tagging site versatility. E

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Bioconjugate Chemistry fixed by 4% paraformaldehyde in PBS (w/v), and incubated with anti-HA-FITC antibody (in 1:500 dilution) (SigmaAldrich, USA) at RT for 2 h. The cells were washed 4 times with PBS and imaged by confocal fluorescent microscope.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00334. Supporting methods, analysis, sequencing results, and data on mass of the synthetic peptide and primers used in cloning (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (852) 3943 6165. Fax: (852) 2603 5057. ORCID

Jiang Xia: 0000-0001-8112-7625 Author Contributions

M.L. and Z.J. performed the experiments and analyzed data; M.Z. and J.X. designed the experiments; J.X. drafted the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from the University Grants Committee of Hong Kong (GRF grants 404413, 14304915, and 14321116, and AoE/M-09/12).

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ABBREVIATIONS Fmoc, 9-fluorenylmenthoxycarbonyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; DMF, N,N-dimethylformamide; DIEA, N,N-diisopropylethylamine; FAM, 5-(and-6)carboxyfluorescein; TAMRA, 5-(and-6)-tetramethylrhodamine; DCM, dichloromethane; TFA, trifluoroacetic acid; EDC, 3(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine; Dap, diamino propionic acid; Mtt, 4-methyltrityl; TIS, triisopropylsilane; IPTG, isopropyl β-D-1-thiogalactopyranoside; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.7b00334 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.7b00334 Bioconjugate Chem. XXXX, XXX, XXX−XXX