Monitoring Conformational Changes in Peroxisome Proliferator

Article pubs.acs.org/jmc

Monitoring Conformational Changes in Peroxisome ProliferatorActivated Receptor α by a Genetically Encoded Photoamino Acid, Cross-Linking, and Mass Spectrometry Rico Schwarz, Dirk Tan̈ zler, Christian H. Ihling, Mathias Q. Müller, Knut Kölbel, and Andrea Sinz* Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, D-06120 Halle/Saale, Germany S Supporting Information *

ABSTRACT: Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction products has evolved into an alternative strategy to structurally resolve protein complexes. We investigated conformational changes in peroxisome proliferator-activated receptor α (PPARα) upon ligand binding. Using E. coli cells with a special tRNA/aminoacyl-tRNA synthetase pair, two PPARα variants were prepared in which Leu-258 or Phe-273 were site-specifically replaced by the genetically encoded photoreactive amino acid p-benzoylphenylalanine (Bpa). PPARα variants were subjected to UV-induced cross-linking, both in the absence and in the presence of ligands. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested and peptides were analyzed by mass spectrometry. The inter-residue distances disclosed by the photochemical cross-links served to monitor conformational changes in PPARα upon agonist and antagonist binding. The data obtained with our strategy emphasize the potential of genetically encoded internal photo-cross-linkers in combination with mass spectrometry as an alternative method to monitor in-solution 3D-protein structures.

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INTRODUCTION Establishing and improving reliable analytical methods for screening target protein−drug interactions are essential for drug discovery. The present study is part of our ongoing research efforts to develop novel strategies based on chemical cross-linking and mass spectrometry (MS) for 3D-structure analysis of target proteins. Cross-linking allows establishing a set of structurally defined interactions by covalently connecting pairs of functional groups within a protein. From the distance information obtained by the chemical cross-links, distance maps can be created within a protein or a protein complex, which serve as basis for deducing low-resolution 3D-structures.1−6 More specifically, conformational changes might be monitored within a target protein upon drug binding.7 The strengths of this strategy comprise the theoretically unlimited size of the protein or protein complex under investigation and the minimal sample requirements in the femtomole to attomole range.1−10 In this work, we extend conventional cross-linking approaches, which are usually based on amine-reactive crosslinkers, by incorporating the genetically encoded photoactivatable amino acid p-benzoylphenylalanine (Bpa) at specific positions into the ligand binding domain (LBD) of peroxisome proliferator-activated receptor α (PPARα) according to the method of Schultz.11 The incorporation of Bpa followed by a mass spectrometric analysis of the created photo-cross-links has been described recently for monitoring the conformation of the ISWI ATPase domain.12 PPARα presents an important drug target, making it an attractive system to evaluate this photochemical cross-linking strategy. The benzophenone group of Bpa is activated by long-wavelength UV light (365 © XXXX American Chemical Society

nm) and can insert into CH and NH groups of various amino acids (Scheme 1).13 This is a distinct advantage over crosslinking strategies with N-hydroxysuccinimide (NHS) esters that mainly react with amine groups of lysines. Clearly, photo-crosslinking with benzophenones is more versatile than cross-linking with NHS esters, although benzophenones have been reported to possess a certain preference for methionines.14,15 The created photo-cross-linked products are identified after enzymatic digestion of PPARα by mass spectrometric analysis of the cross-linked peptides. The distance constraints imposed by the photochemical cross-links serve as “molecular rulers” to map distances within free and ligand-bound PPARα and allow monitoring of conformational changes in PPARα upon ligand binding. PPARs are ligand-activated transcription factors that belong to the nuclear receptor protein family. Three subtypes of PPARs (α, δ (or β, Nuc-1), and γ) have been identified so far.16−18 PPARs are activated by fatty acids and eicosanoids and are targets for antidiabetic and lipid-lowering drugs.19−22 Activated PPARs form heterodimers with the retinoid X receptor (RXR) and bind to specific DNA sequences.23,24 In general, PPARα promotes fatty acid catabolism in the liver and in skeletal muscle while PPARγ regulates fatty acid storage in adipose tissues.25−27 Fibrates have been used since the 1960s for treating hypertriglyceridemia and have recently been shown to be PPARα agonists.21 Received: December 15, 2012

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dx.doi.org/10.1021/jm400446b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 1. Cross-Linking Reaction of the Photoreactive Amino Acid Bpa

amino acid Bpa were thoughtfully selected: Leu-258 is located in the Ω-loop of PPARα, which had been shown to undergo a conformational change upon binding of the antagonist GW6471.7 Phe-273 is located on α-helix 3 of PPARα in direct neighborhood to the flexible Ω-loop. Bpa contains a rather bulky side chain; therefore, one has to consider steric and hydrophobic effects that might influence the protein’s 3Dstructure after Bpa incorporation. Yet CD spectra of Bpa variants and wild type PPARα looked essentially identical (data not shown). In a recent report, fluorescence emission of a pyrene-based PPARα/δ coagonist was measured upon binding of a PPARα variant, in which Leu-258 was exchanged to a tryptophan residue.32 This study suggested the spatial proximity of the Ω-loop and the occupied ligand binding pocket, yet leaving the exact position of residue 258 in the PPARα−ligand complex open to further investigation. Purification of PPARα Variants L258Bpa and F273Bpa. PPARα-Bpa variants were purified in two consecutive chromatographic steps, starting with a nickel affinity purification using the proteins’ C-terminal His-tag (Figure 1A, Supporting Information Figure S1). The subsequent anion exchange chromatography yielded pure protein (Figure 1B, Supporting Information Figure S2). The protein concentration was determined to be ∼4 μM for both PPARα-Bpa variants, corresponding to a protein yield of ∼0.13 mg/g cells. Contaminants in the PPARα-Bpa preparations were identified by peptide fragment fingerprint analysis of selected SDS− PAGE bands (Figure 1) using in-gel digestion with trypsin and analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS (Supporting Information Table S1). The identity of the PPARα variants and the complete incorporation of Bpa at the desired positions were confirmed in the same manner. Identification of Photo-Cross-Links. After purification of PPARα-L258Bpa and PPARα-F273Bpa, we conducted photocross-linking experiments by irradiation with different UV-A doses in the absence and presence of the PPARα ligands GW647128 (antagonist) or GW764729 (agonist). After the photo-cross-linking reaction, PPARα variants were in-gel digested or in-solution digested with trypsin or a mixture of trypsin and GluC (see Experimental Section) and the resulting peptide mixtures were analyzed by nano-HPLC/nano-ESILTQ-Orbitrap-MS/MS. A number of cross-linked products were identified in the presence and in the absence of ligands for both PPARα-L258Bpa (Table 1, Supporting Information Table S2) and PPARα-F273Bpa (Table 2, Supporting Information Table S3). Obviously, Bpa had reacted with a wide range of amino acids. Exemplary MS and MS/MS data are presented for the identification of mixed cross-links between Bpa-258 and Phe-218/Arg-226 (Figure 2). This emphasizes the strength of the tandem MS analysis, as even mixed cross-linked products are clearly discerned. Another cross-link was unambiguously identified between Bpa-258 and Pro-238 (Figure 3), which was assigned based on the b6 ions at m/z 559.4 (triply charged) and m/z 837.8 (doubly charged) and the y15 ion at m/z 967.1 as

In this paper, we study the conformational changes in PPARα upon binding of the antagonist GW6471 (IC50 = 0.24 μM,28 Scheme 2) and the agonist GW7647 (EC50 = 0.006 Scheme 2. Chemical Structure of Antagonist GW6471

Scheme 3. Chemical Structure of Agonist GW7647

μM,29 Scheme 3). A leucine residue (Leu-258) located in the flexible, so-called “Ω-loop” of PPARα’s LBD and a phenylalanine residue (Phe-273) in α-helix 3 were replaced by the photoamino acid Bpa. The photo-cross-linking reaction was induced by UV-A irradiation, and the created distance constraints served to detect conformational changes in PPARα upon GW7647 (agonist) and GW6471 (antagonist) binding. The data obtained with our photo-cross-linking strategy are in agreement with previous structural data of PPARα in the presence and absence of ligands and emphasize the potential of this approach as an alternative method to monitor conformational changes in target proteins.

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RESULTS The crystal structure of PPARα’s LBD has been determined in complex with the antagonist GW6471.28 No structural information is available on how GW7647 influences PPARα conformation. Two Bpa variants of the 32 kDa LBD of PPARα were expressed in E. coli as C-terminally His-tagged constructs using the method developed in the Schultz lab.11,12,30,31 In the following, we will refer to the LBD of PPARα simply as PPARα. The PPARα variants were individually transformed into E. coli cells carrying a Bpa-specific suppressor tRNA and aminoacyl-tRNA synthetase, which allow incorporation of Bpa in place of the introduced TAG stop codon. The amino acids (Leu-258 and Phe-273) that were replaced by the photoreactive B



Article

Table 2. Summary of Cross-Linked Amino Acids Identified in PPARα-F273Bpa after In-Gel Digestion with agonist GW7647

with antagonist GW6471

E212 or A213 I354 or M355 or E356

I210 or Y211 L258

without ligand I210 or Y211 N217 N217 or F218 or N219 A225 I228 M244 or E245 T246 or L247 or C248 T253 or L254 L254 A256 or K257 L258 L258 or V259 or A260 I354 or M355 or E356 A362

F273Bpa). For PPARα-L258Bpa, the cross-links obtained from in-gel digestion of the monomer bands were comparable between free and antagonist-bound protein, while in agonistbound PPARα the number of cross-links was slightly lower and directed toward fewer regions within the protein (Table 1). In PPARα-F273Bpa, on the other hand, the number of cross-links greatly differed between free and ligand-bound protein (Table 2). As Bpa-258 is located on the flexible Ω-loop itself, several cross-links were observed to neighboring residues on the Ωloop in ligand-free and ligand-bound states. In the PPARα-F273Bpa variant, the cross-linking patterns were strikingly different: A principally different behavior was observed in this variant between ligand-free and ligand-bound form, regardless of agonist or antagonist. In antagonist-bound PPARα-F273Bpa, two cross-links were found with Ile-210 or Tyr-211 in α-helix 1 and Leu-258 in the Ω-loop, which point to the same regions to be cross-linked as in free PPARα. In the presence of the agonist, a cross-link between Bpa-273 and amino acids 354−356 is identical to that found in free PPARα. Another cross-link to Glu-212 or Ala-213 in α-helix 1 is in direct neighborhood to amino acid Ile-210 or Tyr-211 (Table 2). In free PPARα only, residues in α-helix 2 (Ala-225, Ile-228) were found to be cross-linked but not in liganded PPARα. For free and antagonist-bound PPARα-F273Bpa, cross-links were found between Bpa-273 and the flexible Ω-loop, while for the agonist-bound form, no cross-link was detected to the Ω-loop.

Figure 1. SDS−PAGE analysis (12% gel) of (A) nickel affinity chromatography (for chromatogram see Supporting Information Figure S1) and (B) anion exchange chromatography (for chromatogram see Supporting Information Figure S2) of PPARα-L258Bpa purification. For protein identification, bands 1−16 were excised, in-gel digested with trypsin, and analyzed by nano-HPLC/nano-ESI-LTQOrbitrap-MS/MS (Supporting Information Table S1): M, protein marker; L, load fraction; T, flow-through fractions; W2 (part A) and A and C (part B), wash fractions; 3-21 and E-K, elution fractions (Supporting Information Figures S1 and S2).

well as b4 and b5 ions. This is the first time that we found a proline to have reacted with Bpa. Interestingly, one fragment ion at m/z 1107.8 pointed to an additional cross-link with Ala233. In a number of cases we were not able to unambiguously identify one specific amino acid residue as cross-linking site by MS/MS (Supporting Information Figure S3). In these cases, all potential reaction sites are given (Tables 1 and 2 and Supporting Information Tables S2 and S3). 3D-Structural Information Derived from Photo-CrossLinks. All cross-links are visualized in the crystal structure (PDB entry 1KKQ) of PPARα (Figure 4 showing PPARα variant L258Bpa and Figure 5 showing PPARα variant

Table 1. Summary of Cross-Linked Amino Acids Identified in PPARα-L258Bpa after In-Gel Digestion with agonist GW7647

with antagonist GW6471

without ligand

L254 V255 A256 or K257 P350 or F351 Y468 E471 H477

Y214 L254 V255 A256 or K257 K257 E267 or A268 or E269 or V270 or R271 or I272 or F273 F359 or D360 or F361 or A362 or M363 or K364 M363 or K364 D466 or M467 D466 or M467 or Y468

N217 A225 A233 P238 L254 V255 V270 A333 P458 or L459 M467 H475 H477

C



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Figure 2. MS and MS/MS analysis of a cross-linked product in PPARα-LBD-L258Bpa (see Table 1): (A) mass spectrum showing a triply charged ion at m/z 773.066; (B, C) fragment ion mass spectrum (MS/MS) of the precursor ion at m/z 773.066. MS/MS data confirmed mixed cross-links of Bpa-258 with (B) Phe-218 and (C) Arg-226. n, deamidated Asn.

to amino acids on the AF2 helix and thus cannot be crosslinked. In summary, our cross-linking results indicate that in both ligand-bound and -free states, similar conformational states are populated in PPARα and that some of them are stabilized upon ligand binding. Apparently, it depends on the nature of the ligand, which conformation is preferred.

Activation function 2 (AF2) helix was found to be crosslinked to Bpa-258 in all states (free and liganded PPARα), while no cross-link was detected between the AF2 helix and Bpa-273. Apparently, the inherent flexibilities of the AF2 helix and the Ω-loop allow cross-links to be created between amino acids on the AF2 helix and Bpa-258. As Bpa-273 is located outside the flexible Ω-loop, it does not come into close distance D



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Figure 3. MS and MS/MS analysis of a cross-linked product in PPARα-LBD-L258Bpa in the absence of ligands (see Table 1): (A) mass spectrum showing a 4+ charged ion at m/z 850.402; (B) fragment ion mass spectrum (MS/MS) of the precursor ion at m/z 850.402. MS/MS data confirmed a cross-link between Bpa-258 and Pro-238. An additional cross-link between Bpa-258 to Ala-233 was confirmed based on the fragment ion at m/z 1107.8. n, deamidated Asn; B, carbamidomethylated Cys.

The number of photo-cross-links found in this study is higher compared to that identified in our previous studies with aminereactive cross-linkers, which, however, had been conducted exclusively with the antagonist GW6471.7 Together, the distance constraints obtained by photo-cross-linking give a more complex picture of the conformations that PPARα can adopt in its free and ligand-bound states than perceived in our previous cross-linking studies. MALDI-TOF Mass Spectrometry of Intact PPARα/ Ligand Complexes. In addition to the cross-links that were found after enzymatic digestion of the protein, we aimed to detect intact photo-cross-linked complexes between PPARαBpa variants with GW6471 and GW7647 by MALDI-TOF mass spectrometry. Interestingly, we observed signals of crosslinked complexes between PPARα-Bpa variants only for the agonist but not for the antagonist (Figure 6). This might originate from different orientations of the ligands in the binding pocket, which could bring the agonist GW7647 in closer spatial proximity to the reactive group of Bpa than the antagonist GW6471. Alternatively, the inherent chemical

properties of both substances (Schemes 2 and 3) indicate that the agonist GW7647 is more susceptible to react with Bpa than the antagonist GW6471. UV-A irradiation of mixtures of pure agonist and antagonist with Bpa and subsequent analysis by ESI-LTQ-Orbitrap mass spectrometry yielded highly intense signals exclusively for the Bpa/agonist adduct (data not shown), which gives a strong hint on the latter explanation. Dimer Formation after Photo-Cross-Linking. For both PPARα-Bpa variants, a certain amount of dimer formation was observed after photo-cross-linking (Supporting Information Figure S4B). Photo-cross-linked PPARα-Bpa variants were subjected to size-exclusion chromatography, showing clearly separated peaks for monomeric and dimeric species (Supporting Information Figure S4A). PPARα-Bpa was identified in both peaks by peptide mass fingerprint analysis. Dimer formation was also visible in SDS−PAGE analysis (Supporting Information Figures S5 and S6). This implies that cross-links identified from in-solution digestion might in fact represent intermolecular cross-linked species of two PPARα monomers. Therefore, we considered cross-linked products originating E



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Figure 4. Photo-cross-links identified in PPARα-L258Bpa (presented in PDB entry 1KKQ) (A) without ligand, (B) with agonist GW7647, and (C) with antagonist GW6471 (magenta): activation function helix 2 (AF2, amino acids 458−467), orange; Ω-loop (amino acids 231−265), yellow; cross-linked amino acids, red; Leu-258 that was exchanged with Bpa, green. In these cases where cross-linked amino acids were not unambiguously identified, amino acids are colored salmon and amino acid numbers are shown in gray.

one single amino acid as a cross-linking site. For these cases where we were not able to assign one amino acid, we present a defined amino acid sequence stretch as a potential cross-linking site (Figures 4 and 5). In summary, conformational changes induced by ligand binding are more pronouncedly observed in the PPARαF273Bpa than in the PPARα-L258Bpa variant. This can be explained by the fact that the Ω-loop itself (where Leu-258 is located) is highly flexible and allows various simultaneously existing conformations to be captured by photochemical crosslinking.

from in-gel digestion of PPARα monomeric bands for deducing 3D-structural information (Tables 1 and 2).

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DISCUSSION Incorporation of the photoreactive amino acid Bpa instead of Leu-258 and Phe-273 in PPARα followed by UV-induced crosslinking yielded a number of defined distance constraints. The identification of a specific amino acid as a cross-link site relies on the identification of indicative fragment ions in MS/MS experiments (Figures 2 and 3). In a number of cases, MS/MS data were not unambiguous, making it impossible to identify F



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Figure 5. Photo-cross-links identified in PPARα-F273Bpa (presented in PDB entry 1KKQ) (A) without ligand, (B) with agonist GW7647, (C) with antagonist GW6471 (magenta): activation function helix 2 (AF2, amino acids 458−467), orange; Ω-loop (amino acids 231−265), yellow; crosslinked amino acids, red; Phe-273 that was exchanged with Bpa, green. In these cases where cross-linked amino acids were not unambiguously identified, amino acids are colored salmon and amino acid numbers are shown in gray.

Some of the cross-links give a hint on major structural changes within PPARα compared to the X-ray structure (PDB entry 1KKQ). Among these are the cross-links between Bpa258 and Asn-217 (in free PPARα)/Tyr-214 (in antagonistbound PPARα) as well as between Bpa-273 and amino acids 210−213 (in free and ligand-bound PPARα). In order to accommodate these cross-links, a large conformational change has to occur, bringing α-helix 1 spatially close to the Bpa residue. It is somewhat puzzling that for the PPARα-F273Bpa

variant, the respective cross-links are found in all three states under investigation (free, with agonist, and with antagonist), whereas for the PPARα-L258Bpa variant, one cross-link to Tyr214 is found in the antagonist-bound state but not in the agonist-bound state (compare Figures 4 and 5). One explanation might be that upon binding of the antagonist GW6471 the Ω-loop (where Bpa258 is located) flips over and brings Tyr-214 in close distance to Bpa-258 in order to be cross-linked. A large conformational change within PPARα G



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Figure 6. MALDI-TOF mass spectra of (A) PPARα-L258Bpa with agonist GW7647, (B) PPARα-L258Bpa with antagonist GW6471, (C) PPARαF273Bpa with agonist GW7647, (D) PPARα-F273Bpa with antagonist GW6471: ∗, degradation product of PPARα; #, catabolite gene activator.

upon GW6471 binding has already been observed in our previous cross-linking experiments with amine-reactive crosslinkers.7 Intriguingly, in free PPARα, Asn-217 is cross-linked to Bpa-258, indicating that this conformation is also present in the absence of ligands. NMR studies on liganded and free PPARα had indicated that the LBD is stabilized upon ligand binding,17 which is in principle confirmed by our photo-cross-linking results. The obtained cross-links are schematically presented in the linearized PPARα amino acid sequences (Figure 7 showing PPARα-L258Bpa variant and Figure 8 showing PPARαF273Bpa variant). From these plots it is readily visible that the cross-links are similar for free and ligand-bound PPARαL258Bpa variant. The plots look highly similar for agonist- and

antagonist-bound PPARα, with the only difference of Tyr-214 to be cross-linked to Bpa-258 in the presence of the antagonist GW6471 (see above and Figure 7). Cross-links between Bpa258 and residues in the AF2 helix are comparable between ligand-free and ligand-bound states. From the cross-links found in the PPARα-L258Bpa variant, a slightly higher flexibility can be deduced in the ligand-free form compared to the ligandbound states. For the PPARα-L273Bpa variant, the plots show a different pattern (Figure 8). In the absence of a ligand, Bpa-273 is crosslinked to a number of amino acids in α-helices 1 and 2 and in the Ω-loop, indicating the presence of various simultaneous conformations. On the other hand, in the presence of ligand (both agonist and antagonist) only a small number of crossH



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Figure 7. Schematic plot of photo-cross-links identified in PPARα-L258Bpa (A) without ligand, (B) with agonist GW7647, (C) with antagonist GW6471. Cross-linked amino acids, which were unambiguously identified by MS/MS data, are presented as solid lines. In these cases where an unambiguous assignment was not possible, potentially cross-linked amino acids are indicated as dotted lines.

structural changes in the AF2 helix, additional Bpa variants will be created.

links were found. This hints that a limited number of conformations are adopted in PPARα, with a few of them being preferentially stabilized in the ligand-bound form, which results in a generally more rigid structure. The fact that some cross-links are found for free and ligandbound PPARα alike gives another hint that PPARα exists in a number of conformations simultaneously and that the nature of the ligand determines which of the coexisting conformations is actually preferred. It has been described that the C-terminal helix 12 (AF2 helix) can transiently adopt a relatively stable active conformation even in the absence of ligand.24 The positioning of this helix influences transcriptional activity via interactions.33 On the basis of the cross-links found in the PPARα-L258Bpa variant (Figures 4 and 7), however, we cannot draw detailed conclusions on a different positioning of the AF2 helix depending on the absence or presence of the two ligands investigated herein. The positions of the amino acids (Leu-258 and Phe-273), which were exchanged by Bpa, do not allow deduction of structural differences in the arrangement of the AF2 helix upon ligand binding. In order to clarify defined

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CONCLUSIONS AND OUTLOOK Incorporation of the photoreactive amino acid Bpa at two defined positions in PPARα followed by UV-induced crosslinking and high-resolution mass spectrometry yielded a number of defined cross-links within PPARα in the absence and presence of the antagonist GW6471 and the agonist GW7647. Our experiments show that the position of the amino acid to be exchanged by Bpa is of great importance. As Bpa is a rather bulky amino acid, we are currently exploring the incorporation of photoreactive amino acids containing diazirines, such as photomethionine and photoleucine34 Conclusively, the data obtained with the presented photocross-linking strategy give 3D-structural information using low amounts of protein and make it attractive for monitoring insolution conformations of target protein/drug complexes.

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EXPERIMENTAL SECTION

Reagents. Water was purified with a TKA X-CAD system (Thermo Fisher Scientific). Nano-HPLC solvents were gradient I



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Figure 8. Schematic plot of photo-cross-links identified in PPARα-F273Bpa (A) without ligand, (B) with agonist GW7647, (C) with antagonist GW6471. Cross-linked amino acids, which were unambiguously identified by MS/MS data, are presented as solid lines. In these cases where an unambiguous assignment was not possible, potentially cross-linked amino acids are indicated as dotted lines. incorporation (see above). Cells were grown at 37 °C in terrific broth (TB) medium in the presence of 30 μg/mL kanamycin and 32 μg/mL chloramphenicol to an OD600 of 0.6−0.8. After the mixture was cooled to 18−20 °C, 0.4% (w/v) glucose, 1% (w/v) sorbitol, and 1% (w/v) saccharose were added. Then 0.1 mM Bpa in 0.5 M NaOH was added to the medium. Concentrations of antibiotics were adjusted before induction was started with 0.2% arabinose (w/v) and 0.1 mM isopropylthio-β-D-galactoside (IPTG). Cells were grown for 12−14 h at 18−20 °C before they were harvested by centrifugation (5000 rpm, 4 °C, Eppendorf 5804R centrifuge, Hamburg, Germany) for 20 min and resuspended in 1 mL/g cells in IMAC-A buffer (20 mM imidazole, 20 mM HEPES, 150 mM NaCl, 10% (v/v) glycerol, pH 8). To the resuspended cells, one tablet of protease inhibitor complete (Roche, Mannheim, Germany), DNase1 (5 μg/mL), RNase A (10 μg/ mL), and 100 mM phenylmethanesulfonyl fluoride (PMSF) were added. After sonication on ice, the lysate was centrifuged at 16000g (4 °C, 60 min). Before chromatographic separation, the supernatant was filtered using Filtropur S 0.2 μm filtration units (Sarstedt, Nümbrecht, Germany). All purification steps were carried out on an Ä KTA FPLC system (GE Healthcare, Munich, Germany) at 8 °C. For nickel affinity chromatography the supernatant was loaded at 0.5 mL/ min onto a 1 mL HisTrap FF column (GE Healthcare) using a 50 mL superloop or 10 mL loop (GE Healthcare). PPARα-L258Bpa and

grade (LiChrosolv Merck, Darmstadt, Germany). GW6471 was purchased from Sigma-Aldrich (Taufkirchen, Germany). GW7647 was obtained from Cayman Chemical Company (Ann Arbor, MI). Trypsin (cleaving C-terminally of lysine and arginine) was obtained from Promega (Mannheim, Germany). GluC (cleaving C-terminally of glutamate and aspartate) was obtained from Roche Diagnostics (Mannheim, Germany). Tryptone, yeast extract, antibiotics, and IPTG were purchased from Roth (Karlsruhe, Germany). MALDI matrices and proteins for MALDI-TOF-MS calibration were obtained from Bruker Daltonik (Bremen, Germany). Iodacetamide, dithiothreitol (DTT), imidazole (Merck, Darmstadt, Germany), and all other chemicals (Sigma-Aldrich, Taufkirchen, Germany) were obtained at the highest available purity. Protein Expression and Purification. Expression and purification of the ligand-binding domain (LBD) of PPARα were essentially carried out as described recently.7,29 The photoactivatable amino acid Bpa was incorporated into PPARα at positions 258 and 273 according to the method described by Schultz et al.11,31 Briefly, pMMCHis PPARαL258Stop or pMMCHis PPARαF273Stop and pEvol-pBpF were co-transformed into E. coli BL21 (DE3). At a later stage, we found expression yields much improved by using pet28A plasmids (optimized for expression of eukaryotic genes in E. coli, Eurofins MWG Operon, Huntsville, AL) that were adapted by us for Bpa J



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PPARα-F273Bpa eluted at a flow rate of 1 mL/min using a two-step elution using 10% (v/v) and 100% (v/v) IMAC-B buffer (500 mM imidazole, 20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 8). PPARα variants eluted at 10% (v/v) IMAC-B buffer (50−60 mM imidazole), and PPARα-containing fractions were pooled and diluted 1:2 (v/v) with AEX-A buffer (100 mM NaCl, 20 mM HEPES, 10% glycerol, pH 8). For the subsequent anion exchange chromatography step, the solution was loaded at a flow rate of 0.5 mL/min onto a 1 mL HiTrap Q XL column (GE Healthcare). Again, a two-step elution with 10% (v/v) and 100% (v/v) AEX-B buffer (500 mM NaCl, 20 mM HEPES, 10% glycerol, pH 8) was carried out. PPARα-L258Bpa and PPARα-F273Bpa were mainly present in the flow-through as well as in the fractions eluting with 10% (v/v) AEX-B buffer (150 mM NaCl). PPARα-containing fractions were pooled, concentrated via Amicon ultracentrifugation units (cutoff 10 kDa; Millipore, Darmstadt, Germany) to 4−6 μM and stored at −20 °C before photo-crosslinking experiments were conducted. Photo-Cross-Linking. PPARα-L258Bpa or PPARα-F273Bpa solution (4 μM) was mixed with the agonist GW7647 in ethanol or the antagonist GW6471 in DMSO to a give a 200-fold molar excess of ligand over the protein. As a control, one sample was mixed with DMSO or ethanol without the addition of ligand. After 30 min at 4 °C, both solutions were irradiated with different doses (4, 6, 8, 12, and 16 J/cm2) of UV-A light (maximum at 365 nm) to activate the photoamino acid Bpa (Scheme 1). Afterward, samples were analyzed by SDS−PAGE or subjected to acetone precipitation followed by insolution tryptic digestion.35 From SDS gels, bands of interest were excised and in-gel digested with trypsin or a mixture of GluC and trypsin according to an existing protocol.36 After enzymatic digestion, the peptide mixtures were immediately analyzed by LC/MS. Nano-HPLC/Nano-ESI-LTQ-Orbitrap-MS/MS Analysis. In-solution and in-gel digestion mixtures were analyzed by LC/MS on an UltiMate nano-HPLC system (LC Packings/Dionex, Idstein, Germany) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoelectrospray ionization (ESI) source (Proxeon). Samples were loaded onto a trapping column (Acclaim PepMap C18, 100 μm × 20 mm, 5 μm, 100 Å, LC Packings) and washed for 15 min with 0.1% TFA at a flow rate of 20 μL/min. Trapped peptides were eluted using a separation column (Acclaim PepMap C18, 75 μm × 250 mm, 3 μm, 100 Å, LC Packings) that had been equilibrated with 100% A (5% acetonitrile, 0.1% formic acid). Peptides were separated with linear gradients from 0% to 40% B (80% acetonitrile, 0.08% formic acid) in 90 min. The column was kept at 30 °C, and the flow rate was 300 nL/ min. During the gradient, online MS data were acquired in datadependent MS/MS mode: Each high-resolution full scan (m/z 300− 2000, resolution 60 000) in the Orbitrap analyzer was followed by five product ion scans (collision-induced dissociation (CID) MS/MS) in the linear ion trap for the five most intense signals of the full scan mass spectrum (isolation window 2.5 Th). Dynamic exclusion (repeat count was 3, exclusion duration 180 s) was enabled to allow detection of less abundant ions. Mass accuracy was set to 3 ppm and 0.8 Da for precursor and fragment ions, respectively. Peptides were identified with the Proteome Discoverer 1.3 (Thermo Fisher Scientific) using Mascot server, version 2.2. Cross-linked products were identified with the in-house software StavroX, version 2.0.6.37 All cross-links were manually evaluated. MALDI-TOF-MS of Intact Proteins. Samples were prepared on a steel target using 1 μL of sinapinic acid (Sigma, Germany) matrix (saturated solution in 90% (v/v) acetonitrile/0.1% (v/v) TFA/1 mM ammonium dihydrogen phosphate) and 2 μL of protein solution after acetone precipitation in 50% (v/v) ACN/0.1% (v/v) TFA. MALDITOF-MS measurements were conducted in the linear and positive ionization modes on an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). Spectra in the mass range m/z 15000−40000 were acquired under the control of FlexControl 3.0 operation software and processed with the FlexAnalysis 3.0 software (Bruker Daltonik).

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ASSOCIATED CONTENT

S Supporting Information *

Purification of PPARα-Bpa variants, MS and MS/MS analyses of a PPARα-L258Bpa cross-link, analysis of photo-cross-linked PPARα-Bpa dimer (size-exclusion chromatography and SDS− PAGE), listing of contaminating proteins in PPARα preparations, and detailed mass spectrometric data (precursor ion masses, fragment ions, amino acid sequences) for all crosslinked products described herein. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-345-5525170. Fax: +49-345-5527026. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This paper is dedicated to the late Otto Sinz. A.S. acknowledges support from the DFG (Projects Si 867/15-1 and 16-1), the BMBF (Grant ProNet-T3), and the Land Sachsen-Anhalt. The authors thank Peter Schultz for the donation of plasmids.

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ABBREVIATIONS USED AEX, anion exchange chromatography; AF2, activation function 2; Bpa, p-benzoylphenylalanine; CID, collision-induced dissociation; Da, Dalton; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; IMAC, immobilized metal ion chromatography; IPTG, isopropylthio-β-D-galactoside; LB, lysogenic broth; LTQ, linear ion trap (Thermo Fisher Scientific); NHS, N-hydroxysuccinimide; PMSF, phenylmethanesulfonyl fluoride; PPARα, peroxisome proliferator-activated receptor α; RXR, retinoid X receptor; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; TB, terrific broth; TCEP, tris(2-carboxyethyl)phosphine; XL, cross-linker/ cross-link

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REFERENCES

(1) Young, M. M.; Tang, N.; Hempel, J. C.; Oshiro, C. M.; Taylor, E. W.; Kuntz, I. D.; Gibson, B. W.; Dollinger, G. High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802−5806. (2) Sinz, A. Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein−protein interactions. Mass Spectrom. Rev. 2006, 25, 663−682. (3) Sinz, A. Isotope-labeled photoaffinity reagents and mass spectrometry to identify protein−ligand interactions. Angew. Chem., Int. Ed. 2006, 46, 660−662. (4) Rappsilber, J. The beginning of a beautiful friendship: crosslinking/mass spectrometry and modelling of proteins and multiprotein complexes. J. Struct. Biol. 2011, 173, 530−540. (5) Leitner, A.; Walzthoeni, T.; Kahraman, A.; Herzog, F.; Rinner, O.; Beck, M.; Aebersold, R. Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics. Mol. Cell. Proteomics. 2010, 9, 1634−1649. (6) Fabris, D.; Yu, E. T. Elucidating the higher-order structure of biopolymers by structural probing and mass spectrometry: MS3D. J. Mass Spectrom. 2010, 45, 841−860. (7) Müller, M. Q.; de Koning, L. J.; Schmidt, A.; Ihling, C.; Syha, Y.; Rau, O.; Mechtler, K.; Schubert-Zsilavecz, M.; Sinz, A. An innovative method to study target protein−drug interactions by mass spectrometry. J. Med. Chem. 2009, 52, 2875−2879.

K



Article

(8) Kalisman, N.; Adams, C. M.; Levitt, M. Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 2884−2889. (9) Herzog, F.; Kahraman, A.; Boehringer, D.; Mak, R.; Bracher, A.; Walzthoeni, T.; Leitner, A.; Beck, M.; Hartl, F. U.; Ban, N.; Malmström, L.; Aebersold, R. Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry. Science 2012, 337, 1348−1352. (10) Chen, Z. A.; Jawhari, A.; Fischer, L.; Buchen, C.; Tahir, S.; Kamenski, T.; Rasmussen, M.; Lariviere, L.; Bukowski-Wills, J. C.; Nilges, M.; Cramer, P.; Rappsilber, J. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 2010, 29, 717−726. (11) Xie, J.; Schultz, P. G. A chemical toolkit for proteinsan expanded genetic code. Nat. Rev. Mol. Cell Biol. 2006, 7, 775−782. (12) Forné, I.; Ludwigsen, J.; Imhof, A.; Becker, P. B.; MuellerPlanitz, F. Probing the conformation of the ISWI ATPase domain with genetically encoded photoreactive crosslinkers and mass spectrometry. Mol. Cell. Proteomics 2012, 11 (4), M111.012088. (13) Dimova, K.; Kalkhof, S.; Pottratz, I.; Ihling, C.; RodriguezCastaneda, F.; Liepold, T.; Griesinger, C.; Brose, N.; Sinz, A.; Jahn, O. Structural insights into the calmodulin-Munc13 interaction obtained by cross-linking and mass spectrometry. Biochemistry 2009, 48, 5908− 5921. (14) Krauth, F.; Ihling, C. H.; Rü ttinger, H. H.; Sinz, A. Heterobifunctional isotope-labeled amine-reactive photo-cross-linker for structural investigation of proteins by matrix-assisted laser desorption/ionization tandem time-of-flight and electrospray ionization LTQ-Orbitrap mass spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 2811−2818. (15) Wittelsberger, A.; Thomas, B. E.; Mierke, D. F.; Rosenblatt, M. Methionine acts as a “magnet” in photoaffinity crosslinking experiments. FEBS Lett. 2006, 580, 1872−1876. (16) Michalik, L.; Wahli, W. Peroxisome proliferator-activated receptors: three isotypes for a multitude of functions. Curr. Opin. Biotechnol. 1999, 10, 564−570. (17) Zoete, V.; Grosdidier, A.; Michielin, O. Peroxisome proliferatoractivated receptor structures: ligand specificity, molecular switch and interactions with regulators. Biochim. Biophys. Acta 2007, 1771, 915− 925. (18) Xu, H. E.; Lambert, M. H.; Montana, V. G.; Plunket, K. D.; Moore, L. B.; Collins, J. L.; Oplinger, J. A.; Kliewer, S. A.; Gampe, R. T.; McKee, D. D.; Moore, J. T.; Willson, T. M. Structural determinants of ligand binding selectivity between the peroxisome proliferatoractivated receptors. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13919− 13924. (19) Plutzky, J. The potential role of peroxisome proliferatoractivated receptors on inflammation in type 2 diabetes mellitus and atherosclerosis. Am. J. Cardiol. 2003, 92, 34J−41J. (20) Evans, R. M.; Barish, G. D.; Wang, Y. PPARs and the complex journey to obesity. Nat. Med. 2004, 10, 355−361. (21) Issemann, I.; Prince, R. A.; Tugwood, J. D.; Green, S. The peroxisome proliferator-activated receptor:retinoid X receptor heterodimer is activated by fatty acids and fibrate hypolipidaemic drugs. J. Mol. Endocrinol. 1993, 11, 37−47. (22) Berger, J. P.; Akiyama, T. E.; Meinke, P. T. PPARs: therapeutic targets for metabolic disease. Trends Pharmacol. Sci. 2005, 26, 244− 251. (23) Qi, C.; Zhu, Y.; Reddy, J. K. Peroxisome proliferator-activated receptors, coactivators, and downstream targets. Cell Biochem. Biophys. 2000, 32, 187−204. (24) Michalik, L.; Zoete, V.; Krey, G.; Grosdidier, A.; Gelman, L.; Chodanowski, P.; Feige, J. N.; Desvergne, B.; Wahli, W.; Michielin, O. Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior. J. Biol. Chem. 2007, 282, 9666−9677.

(25) Desvergne, B.; Wahli, W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev. 1999, 20, 649− 688. (26) Rau, O.; Syha, Y.; Zettl, H.; Kock, M.; Bock, A.; SchubertZsilavecz, M. Alpha-alkyl substituted pirinixic acid derivatives as potent dual agonists of the peroxisome proliferator activated receptor alpha and gamma. Arch. Pharm. 2008, 341, 191−195. (27) Cronet, P.; Petersen, J. F.; Folmer, R.; Blomberg, N.; Sjöblom, K.; Karlsson, U.; Lindstedt, E. L.; Bamberg, K. Structure of the PPARalpha and -gamma ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure 2001, 9, 699−706. (28) Xu, H. E.; Stanley, T. B.; Montana, V. G.; Lambert, M. H.; Shearer, B. G.; Cobb, J. E.; McKee, D. D.; Galardi, C. M.; Plunket, K. D.; Nolte, R. T.; Parks, D. J.; Moore, J. T.; Kliewer, S. A.; Willson, T. M.; Stimmel, J. B. Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARα. Nature 2002, 415, 813−817. (29) Brown, P. J.; Stuart, L. W.; Hurley, K. P.; Lewis, M. C.; Winegar, D. A.; Wilson, J. G.; Wilkinson, W. O.; Ittoop, O. R.; Willson, T. M. Identification of a subtype selective human PPARα agonist through parallel-array synthesis. Bioorg. Med. Chem. Lett. 2001, 11, 1225−1227. (30) Müller, M. Q.; Roth, C.; Sträter, N.; Sinz, A. Expression and purification of the ligand-binding domain of peroxisome proliferatoractivated receptor alpha (PPARα). Protein Expression Purif. 2008, 62, 185−189. (31) Ryu, Y.; Schultz, P. G. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat. Methods 2006, 3, 263−265. (32) Ban, S.; Oyama, T.; Kasuga, J.; Ohgane, K.; Nishio, Y.; Morikawa, K.; Hashimoto, Y.; Miyachi, H. Bidirectional fluorescence properties of a pyrene-based peroxisome proliferator-actived receptor (PPAR) α/δ dual agonist. Bioorg. Med. Chem. 2012, 20, 3460−3464. (33) Velkov, T.; Rimmer, K. A.; Headey, S. J. Ligand-enhanced expression and in-cell assay of human peroxisome proliferatoractivated receptor alpha ligand binding domain. Protein Expression Purif. 2010, 70, 260−269. (34) Suchanek, M.; Radzikowska, A.; Thiele, C. Photo-leucine and photo-methionine allow identification of protein−protein interactions in living cells. Nat. Methods 2005, 4, 261−267. (35) Kalkhof, S.; Haehn, S.; Paulsson, M.; Smyth, N.; Meiler, J.; Sinz, A. Computational modeling of laminin N-terminal domains using sparse distance constraints from disulfide bonds and chemical crosslinking. Proteins 2010, 78, 3409−3427. (36) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006, 1, 2856−2860. (37) Götze, M.; Pettelkau, J.; Schaks, S.; Bosse, K.; Ihling, C. H.; Krauth, F.; Fritzsche, R.; Kühn, U.; Sinz, A. StavroXa software for analyzing crosslinked products in protein interactions. J. Am. Soc. Mass Spectrom. 2012, 23, 76−87.

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