Studying Weak and Dynamic Interactions of Posttranslationally

Letters pubs.acs.org/acschemicalbiology

Studying Weak and Dynamic Interactions of Posttranslationally Modified Proteins using Expressed Protein Ligation Konstantinos Tripsianes,†,‡,§,⊥ Nam K. Chu,∥,⊥ Anders Friberg,†,‡ Michael Sattler,*,†,‡ and Christian F. W. Becker∥,* †

Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany Center for Integrated Protein Science Munich and Chair of Biomolecular NMR, TU München, Lichtenbergstr. 4, 85747 Garching, Germany § Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic ∥ Institute of Biological Chemistry, University of Vienna, Währingerstr. 38, 1090 Vienna, Austria ‡

S Supporting Information *

ABSTRACT: Many cellular processes are regulated by posttranslational modifications that are recognized by specific domains in protein binding partners. These interactions are often weak, thus allowing a highly dynamic and combinatorial regulatory network of protein−protein interactions. We report an efficient strategy that overcomes challenges in structural analysis of such a weak transient interaction between the Tudor domain of the Survival of Motor Neuron (SMN) protein and symmetrically dimethylated arginine (sDMA). The posttranslational modification is chemically introduced and covalently linked to the effector module by a one-pot expressed protein ligation (EPL) procedure also enabling segmental incorporation of NMR-active isotopes for structural analysis. Covalent coupling of the two interacting moieties shifts the equilibrium to the bound state, and stoichiometric interactions are formed even for low affinity interactions. Our approach should enable the structural analysis of weak interactions by NMR or X-ray crystallography to better understand the role of posttranslational modifications in dynamic biological processes.

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enables differential isotope-labeling of the protein and the ligand peptide to support efficient NMR-based structural analysis. We show that by linking the PTM-ligand with its weakly interacting protein the bound state is fully populated, allowing structural characterization of low affinity complexes, which otherwise would require a large excess of one of the interaction partners (Figure 1a). We demonstrate the utility of this approach with recognition of symmetrically dimethylated arginine (sDMA) by the SMN Tudor domain.

osttranslational modifications (PTMs) of proteins play essential roles in the regulation of various cellular functions, including the (epigenetic) control of gene expression at the level of chromatin, splicing, and signal transduction processes.1,2 In cells, PTM-mediated protein−protein interactions depend on a combination of the intrinsic binding affinities and kinetics, local concentrations of the interacting partners, the cellular context, and the presence of other interacting proteins.3 Moreover, the recognition of posttranslational modifications often involves transient and dynamic interactions,4 which render structural analysis challenging as weak affinities and/or dynamics inhibit crystallization and exchange-induced line broadening can strongly impair the quality of NMR data. Here, we report an efficient strategy that overcomes challenges in structural analysis of such weak interactions involving posttranslational modifications. It is well established that covalent coupling of two weakly interacting moieties shifts the equilibrium to the bound state and stoichiometric interactions.5−9 However, covalent coupling of a posttranslationally modified peptide cannot be achieved genetically using recombinant protein expression. We therefore propose to covalently link the PTM ligand peptide to its protein binding partner by expressed protein ligation (EPL).10−14 EPL also © 2013 American Chemical Society

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RESULTS AND DISCUSSION

SMN associates with proteins carrying sDMA modifications and serves as a platform for the assembly of ribonucleoprotein complexes.15−17 sDMA is recognized by the Tudor domain of SMN that forms a small ∼60 residue β-sheet fold. A characteristic feature of the Tudor structure is an aromatic cage that envelops the methylated guanidino group and mediates cation−π interactions without any additional specific contacts.18 Thus, the interaction is of moderate strength (KD ≈ 0.5 mM), yet specific. Previous attempts to obtain high Received: September 20, 2013 Accepted: December 3, 2013 Published: December 3, 2013 347

dx.doi.org/10.1021/cb400723j | ACS Chem. Biol. 2014, 9, 347−352

ACS Chemical Biology

Letters

crystallization and induces detrimental exchange broadening in NMR spectra. Covalently linking both binding partners can overcome these problems by inducing an increased local concentration with a single ligand site. Thereby, the entropic cost of binding is lowered and the population of the bound state increased (Figure 1a).9 On the basis of available structures of the free SMN Tudor domain,19,20 we designed a fusion protein where the sDMA moiety is linked to the Tudor domain using polyglycine linkers of variable length (Supplementary Figure S1). The site-specific attachment of a small molecule to a much larger protein can be extremely challenging.21 In the case of the SMN Tudor domain, C-terminal attachment appears to be the ideal approach because the known sDMA binding site should be easily accessible via a short linker (Figure 1b). Linker design is of paramount importance in this case to balance the entropic and enthalpic contributions to the binding event. For example, the linker design must provide sufficient flexibility and allow tight control over the attachment point.22 For these reasons we propose the use of EPL. EPL works via intein-mediated introduction of a C-terminal α-thioester into SMN Tudor that subsequently reacts chemoselectively with an sDMA-containing peptide harboring an N-terminal cysteine residue that is required for the ligation reaction.10,23 The poly glycine linker was chosen to simplify peptide synthesis but also considering the fact that native peptide sequences that contain methylated arginine residues and interact with Tudor domains are typically glycine-rich.15,16 This strategy also allows straightforward isotope-labeling of the recombinantly produced SMN Tudor for NMR studies (Figure 1b). Initial efforts to isolate the SMN Tudor with a C-terminal αthioester for ligation reactions were not successful. Cleavage of a Tudor domain-Mxe GyrA intein fusion protein with an excess of mercaptoethanesulfonate (MESNa) resulted in all cases in a lower mass than expected for the Tudor domain cleavage product. The observed mass of 7758 Da corresponds to the Tudor domain without the α-thioester moiety and an additional loss of 18 Da (Supplementary Figure S2a,b). Such a cleavage product could be formed if an intermediate thioester is attacked by an internal cysteine thiol group to give an intramolecular thioester bond (thiolactone). However, this assumption could not be confirmed by experiments designed to resolve such an internal thioester with an excess of thiols such as MESNa, MPAA or ethanethiol (data not shown). The observed product proved stable under all tested conditions, even in the presence of high concentrations of guanidinium hydrochloride (6 M). Also native chemical ligation reactions with cysteine or different peptides containing unprotected N-terminal cysteine residues did not affect the molar mass of the Tudor domain after cleavage and no ligation was observed. Presumably, the formed thiolactone is highly stable, consistent with previous reports using synthetically derived peptides in native chemical ligation reactions, which indicated that the stability of such thiolactones is highly sequence dependent.24−26 Less conceivable is the formation of an intramolecular ester with a side chain hydroxyl group. However, the available data cannot exclude such a lactone formation. To overcome this challenge that prevented a straightforward EPL reaction, a one-pot procedure was developed in which preincubation of the Tudor domain-intein fusion construct and the respective sDMA containing peptide was followed by the addition of an excess of ethanethiol. These conditions gave the ligation product in high yields ranging from 77% to quantitative

Figure 1. Covalent coupling of two interacting moieties. (a) Effect on the stability of weak transient interactions by covalently linking binding partners. (b) Ligation strategy for the attachment of a posttranslationally modified peptide to a recognition domain, allowing differential labeling of protein and ligand. The SMN Tudor domain is expressed in fusion with a C-terminal Mxe-intein and a His6-tag. Direct cleavage of this construct by addition of thiol-containing reagents such as MESNa does not lead to the desired Tudor domain thioester. Based on mass spectrometry results, formation of a highly stable thiolactone is the most likely explanation here. In situ addition of sDMA containing cysteine peptides and ethanethiol gives the desired NCL product that can be analyzed by mass spectrometry and NMR.

resolution structures of the Tudor domain bound to sDMA have relied on the addition of a large excess of the free amino acid at high Tudor protein concentration.18 However, such high concentrations (in the millimolar range) of both protein and ligand can often not be achieved. Another feature of the Tudor−sDMA interactions is that the natural PTM protein ligands of the Tudor domain contain multiple sDMA residues in an unstructured polypeptide chain and employ avidity and increased local concentration effects to achieve enhanced binding affinity. However, the substantial inherent dynamics associated with such multisite ligand interactions has inhibited 348



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Figure 2. NMR analysis of Tudor−sDMA interaction with a covalently linked sDMA moiety. (a) Overlay of 1H,15N HSQC spectra of SMN Tudor ligated with sDMA (linker length 1, 2, 4, or 8 glycine residues) and SMN Tudor saturated with excess of sDMA. Some amide signals that experience additional chemical shift changes due to the linker are indicated. Notice that the effect is attenuated as the linker length increases. (b) Weighted amide chemical shift differences between ligated proteins and fully bound Tudor SMN in excess of sDMA. The inset shows the Tudor-sDMA structure with the C-terminal residues and linker depicted schematically. The linker-induced perturbations higher than the cutoff value are mapped onto the Tudor structure with nitrogen atoms as spheres and colored according to the degree of chemical shift difference from yellow to red (gradient bar at the right side). (c) 1H,13C correlations for aromatic protons of free SMN Tudor, SMN Tudor saturated with excess of sDMA, and SMN Tudor ligated to sDMA with 8 glycines. Chemical shift perturbations due to sDMA binding are indicated with arrows. (d) NOE correlations between the SMN Tudor (ω2, aromatic proton frequencies) and sDMA (ω1, proton frequencies) for the ligated sample where the sDMA is linked by 8 glycines to SMN Tudor (yellow) or when sDMA is saturated with excess of SMN Tudor (black). sDMA chemical shifts are annotated. Some differences on sDMA chemical shifts may be attributed to the presence of linker residues that were not present when sDMA was studied in complex with excess of SMN Tudor.

Notably, the one-pot cleavage and ligation procedure provided direct access to sDMA-ligated SMN Tudor with different linker lengths between the sDMA ligand and the Cterminus of the Tudor domain. Based on our assumption that prebinding orients the sDMA peptide for native chemical ligation, reaction kinetics could provide information about the ideal linker length. Quantification of the ligation yields after 24 h provides a crude measure that linkers with four or more glycine residues are best suitable for providing the necessary flexibility to sDMA to reach its binding pocket while being covalently linked to the Tudor domain (Supplementary Figure S3). Even though formation of a highly stable thiolactone is sequence dependent and not expected to occur in many other cases, prepositioning of the posttranslationally modified peptide

conversion. The resulting Tudor domains covalently linked to the sDMA-containing peptides were separated from His-tagged intein and remaining Tudor domain intein fusion construct by affinity chromatography. The Tudor domains C-terminally linked to sDMA-containing peptides of variable length were analyzed by LC-MS under native and denaturing conditions (Supplementary Figure S2c,d,e). We surmise that the success of this one-pot procedure is based on the preformed sDMA-Tudor domain complex. Upon addition of ethanethiol the fusion protein is cleaved and the Nterminal cysteine residue within the sDMA-peptide is well positioned for transesterification with the newly formed αthioester. This prepositioning prevents the reactions that led to formation of the product found upon direct treatment of Tudor domain-intein with thiols. 349



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tion, oligomerization, and localization.27−29 Special care, however, has to be taken when designing the linker to ensure that it does not interfere with native protein−ligand interactions. An alternative approach for selective protein modifications would be the incorporation of a PTM-carrying amino acid based on genetic code extension.30,31 However, although such an approach would allow the selective incorporation of PTMs in any given position, it would not enable the preparation of a segmentally isotope-labeled sample, i.e., where the recognition domain could be 13C,15N-labeled and the PTM-modified ligand peptide unlabeled (Figure 1b). The strategy proposed here will be applicable for studying the interaction of protein domains that recognize peptide motifs in binding partners and especially useful to study interactions with peptides that carry posttranslational modifications and are thus not easily accessible by recombinant protein expression. The ligation strategy has many advantages: (1) it allows the controlled and unambiguous introduction of specific modifications in the ligands by chemical synthesis, (2) it enables differential isotope labeling of protein and ligand regions for NMR structural analysis, (3) it significantly stabilizes low affinity interactions as often found with multiligand motifs that employ avidity in binding to a single binding pocket, and (4) stoichiometric interactions are obtained even for weakly interacting (peptide) ligands at reasonable protein concentrations suitable for structural and biophysical studies. The approach thus overcomes current limitations32 in structural studies of protein−protein interactions involving posttranslationally modified peptides and will be of great utility for studying the roles of such interactions in cellular processes.

should generally facilitate ligation reactions and provide conclusive information about choosing a suitable linker length. A detailed analysis of the interaction between SMN Tudor and sDMA linked via 1, 2, 4, and 8 glycine residues was carried out by NMR. For this, we prepared fusion proteins using 15 13 N, C-labeled Tudor domain ligated to sDMA with different linker lengths. For each protein we recorded a 1H,15N HSQC spectrum and compared this with a corresponding spectrum of SMN Tudor when saturated with excess of free sDMA. The binding kinetic is fast on the NMR chemical shift time scale, consistent with the weak binding affinity. Depending on the concentration of the free Tudor SMN used, saturation requires 5−20 molar excess of the sDMA ligand.18 In contrast, for all ligated proteins the amide chemical shifts are similar to the fully bound form of the unligated Tudor SMN (Figure 2a). This confirms that the fully bound form of SMN Tudor is already observed when ligated to sDMA at a 1:1 ratio of protein and ligand, independent of the protein concentration. A careful inspection of the NMR spectra shows that all ligated proteins reproduce faithfully the chemical shift perturbations (CSPs) characteristic of sDMA binding. However, additional CSPs map on one side of the Tudor structure (Figure 2). The additional CSPs presumably reflect that the linker designed is too short and imposes a strain on the SMN Tudor structure to allow access of the sDMA moiety to the binding pocket (Figure 2a,b). Interestingly, there is an almost linear relationship between the additional CSPs observed and the linker length (Supplementary Figure S4). This indicates that with increasing linker length, correspondingly less strain is sensed. The ligated protein with an 8-glycine linker is most “relaxed”. In fact, all chemical shifts of this protein are highly similar to the unligated Tudor SMN when saturated with excess of sDMA ligand (Figure 2c, Supplementary Figure S5), indicating that the linker design is optimal as the fully bound state is observed without causing strain or nonspecific linker contacts to the Tudor domain. To demonstrate the utility of our ligation approach toward structural analysis, we recorded filtered NOESY experiments on 13 15 C, N-labeled Tudor domain ligated to unlabeled (Gly)8sDMA. These experiments are compared to NMR data obtained for the fully bound form of the unlabeled sDMA in the presence of an excess of SMN Tudor.18 The NMR signals of the sDMA side chain experience strong upfield shifts due to ring current effects induced by the Tudor aromatic cage, consistent with the structure of the Tudor-sDMA complex. Notably, the NOE effects observed between Tudor SMN and sDMA when covalently linked closely resemble those previously reported, but at substantially reduced concentrations and stoichiometry. The majority of the correlations originate from aromatic Tudor residues that constitute the binding pocket, and the proton frequencies of the ligated sDMA are highly comparable to the ones corresponding to its fully bound state when saturated with excess of Tudor SMN (Figure 2d). The NMR data demonstrate that a weak interaction such as Tudor-sDMA, which is also highly dynamic due to avidity effects with multiple sDMA modifications in the PTM binding partners,18 is substantially enhanced by covalently linking the binding partners. Thereby, interactions that are challenging to structural studies due to their weak and dynamic nature become amenable to structural and biophysical techniques including NMR spectroscopy and X-ray crystallography. The method is also applicable for the attachment of chemical probes, i.e., fluorophores and spin labels, for analyzing protein conforma-

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METHODS

Peptide Synthesis. Fmoc-based solid-phase peptide synthesis was used to prepare peptides containing a symmetrically dimethylated arginine (sDMA) residue. Briefly, 0.2 mmol of Fmoc-Glycine-Wang resin (Novabiochem) was swollen in DMF for 2 h. Fmoc deprotection was achieved by treatment with 20% (v/v) piperidine in DMF for 3 and 7 min, and 2.5 equiv of Fmoc-sDMA was activated with 2.38 equiv of 0.5 M HBTU in DMF and 5 equiv DIEA and added to the resin for 30 min. Peptides containing 1, 2, 4, and 8 glycine residues between the N-terminal cysteine and sDMA were prepared. Each step was followed by 1-min flow-washes with DMF to remove excess reagents. Before cleavage the peptide-resins were vigorously washed with DCM and dried under vacuum. A mixture of 2.5% H 2 O and 2.5% triisisopropylsilane (TIS) in TFA was applied for cleavage over 3 h. Crude products were subsequently precipitated with cooled diethyl ether, dissolved in acetonitrile/water mixtures (1:1, containing 0.1% TFA), and lyophilized. All peptides were purified by RP-HPLC using a C8 RP-column (Vydac) and a linear gradient from 2% to 40% of acetonitrile in H2O (containing 0.08% and 0.1% TFA, respectively) for 60 min. Protein Expression and Purification. The DNA fragment encoding for SMN Tudor (aa 81−149) was PCR amplified and cloned into a modified pTXB3 vector (New England Biolabs) containing the GyrA mini-intein and a chitin-binding domain (CBD) with an additional 6×His tag between them using NcoI and SpeI restriction sites. Thus, a C-terminal intein fusion protein was obtained. For normal expression the resulting plasmid pTXB3-SMN was transformed into E. coli strain BL21(DE3)RIL. For expression of isotope-labeled SMN, BL21(DE3)RIL cells were inoculated overnight at 37 °C in 10 mL of M9 minimum medium containing 13C-glucose and 15N-ammonium chloride and 0.1 mg mL‑1 of ampicillin and 0.03 mg mL‑1 of chloramphenicol. The entire medium was transferred into 1 L of warm (37 °C) M9 minimum medium and grown at 37 °C until the absorption at 600 nm reached 0.6. Expression was induced by 350



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addition of 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG), and cultures were grown at 20 °C overnight. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole), and lysed with a cell disruptor (Basic Z, Constant Cell Disruption System). The lysate was centrifuged at 50,000g for 30 min at 4 °C, and the supernatant was loaded on a Ni-NTA column previously equilibrated with lysis buffer. The column was washed with 5 column volumes of lysis buffer, and SMN-Intein-6×His was eluted with a buffer containing 50 mM TrisHCl, pH 7.5, 300 mM NaCl, and 330 mM imidazole. The eluate fractions were loaded on a Superdex 75 (16/60) column equilibrated with 50 mM Tri-HCl, pH 7.5, 500 mM NaCl. Pure fractions were identified by SDS-PAGE and pooled for the next steps. One-Pot Expressed Protein Ligation (EPL). Purified SMNIntein-6×His protein (100 μM) was mixed with sDMA peptide (500 μM) in 500 μL of 50 mM Tris-HCl at pH 8.0 and 2% ethanethiol. The EPL reactions were incubated at RT for up to 24 h, and 12 mM TCEP was subsequently added to keep the cysteine residues reduced. Crude ligation products were purified by Ni-NTA chromatography. The flowthrough containing the ligation product without a His-tag (SMNsDMA proteins) was loaded on a Superdex 75 (16/60) column and separated from excess sDMA peptides using a buffer consisting of 20 mM NaPi, pH 6.8, 100 mM NaCl, and 0.02% NaN3. Ligation yields were calculated on the basis of relative intensities in MALDI mass spectra (Supplementary Figure S3). SMN Tudor ligated to a peptide with 1 glycine residue was obtained with a relative yield of 77%, a peptide with 2 glycine residues gave 87%, a peptide with 4 glycine residues gave 98%, and the peptide with 8 glycine residues gave quantitative ligation yields. In all cases a minimum of 3.0 mg of SMN Tudor covalently linked to the sDMA was obtained. NMR Spectroscopy. NMR measurements were carried out at 298 K on a Bruker Avance III 750-MHz spectrometer equipped with a TXI probe head. For each ligated protein, the concentration was 200 μM in a buffer containing 20 mM NaPi, pH 6.8, 100 mM NaCl, 2 mM deuterated DTT, and 7% (v/v) 2H2O for the lock.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary figures with analytical data on peptides and proteins as well as additional NMR data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.T. acknowledges support by the Alexander von Humboldt foundation. This work was supported by the European Commission, FP7 NMI3 (no. 226507 to M.S.) and the Deutsche Forschungsgemeinschaft, GRK1721 (M.S.), SFB1035 (M.S.), BE3270 (C.F.W.B.). We thank M. Brehs and K. Baeuml for help with MALDI-MS measurements and peptide synthesis and D. Garg for initial molecular dynamics simulations of ligated SMN Tudor.

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REFERENCES

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Studying Weak and Dynamic Interactions of Posttranslationally

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