Mapping Cell Envelope and Periplasm Protein Interactions of

Article pubs.acs.org/jpr

Mapping Cell Envelope and Periplasm Protein Interactions of Escherichia coli Respiratory Formate Dehydrogenases by Chemical Cross-Linking and Mass Spectrometry Michael Zorn,† Christian H. Ihling,† Ralph Golbik,‡ R. Gary Sawers,§ and Andrea Sinz*,† †

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany ‡ Institute of Biochemistry and §Department of Microbiology, Institute of Biology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany S Supporting Information *

ABSTRACT: During anaerobic growth Escherichia coli synthesizes two large, highly homologous respiratory formate dehydrogenases (Fdh’s), Fdh-N and Fdh-O, which are associated with the inner membrane but have their respective active site located within the periplasm. The Fdh-N enzyme extends 90 Å into the periplasmic compartment, which in E. coli ranges between 100 and 150 Å from the inner to the outer membrane leaflet. To date, little is known about the interaction partners of Fdh-N and Fdh-O in the periplasmic space that might be involved in stabilizing these enzymes after maturation and translocation across the cytoplasmic membrane has occurred. To address this question, we performed chemical cross-linking in combination with mass spectrometry. We present for the first time the identification of cell envelope interaction partners of Fdh-N and -O from anaerobically grown E. coli using a heterobifunctional amine/photoreactive cross-linker followed by mass spectrometric analysis of the cross-linked products. We additionally mapped the interface regions within the Fdh/protein complexes for four selected Fdh-binding partners, the chaperone Skp, the L,D-transpeptidase ErfK, OppA, and TolB. Our work yields first structural and functional insights into the mechanisms that support the postmaturation of the multisubunit enzymes Fdh-N and Fdh-O in the periplasm of E. coli. KEYWORDS: formate dehydrogenases, protein−protein interactions, chemical cross-linking, photoreactive cross-linker, mass spectrometry

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INTRODUCTION The Gram-negative facultative anaerobic γ-proteobacterium Escherichia coli exhibits a modular respiratory chain capable of using a number of electron donors and acceptors. One of these donors is formate, which is generated during anaerobic growth on glucose and is derived from pyruvate by pyruvate formatelyase (PflB).1,2 E. coli synthesizes three formate dehydrogenases (Fdh’s) that are capable of oxidizing formate. One enzyme, Fdh-H, couples formate disproportionation to hydrogen and CO2 and is part of the cytoplasmically oriented formate hydrogenlyase complex.1,2 The other two enzymes are highly similar to each other and are called Fdh-N and Fdh-O to indicate their respective induction after growth with nitrate and oxygen.3,4 Both are energy-conserving, membrane-associated enzymes with their respective active site located facing the periplasm.5 Limited information is available about Fdh-O, although it shows formate oxidase activity during aerobic growth6,7 and is also synthesized during nitrate respiration.8,9 Considerably more is known, however, about the nitrateinducible Fdh-N, which is a large “mushroom-like” 500 kDa protein complex.10 The enzyme is a trimer of trimers that extends 90 Å into the periplasm and has a diameter of 125 Å. © 2014 American Chemical Society

Fdh-N forms a respiratory chain with the cytoplasmically oriented nitrate reductase (Nar) during anaerobic nitrate respiration.4,11−13 Structural analyses of both Fdh-N and Nar enzymes4,14,15 have helped to provide a molecular framework explaining Mitchell’s redox loop theory of chemiosmosis.16,17 The Fdh-N and Fdh-O isoenzymes belong to the molybdopterin-containing oxidoreductase family.2,4 Both enzymes are composed of three subunits. The α-subunit (FdnG/ FdoG) contains the catalytically active selenocysteine, an iron− sulfur [4Fe-4S] cluster, as well as two bis-MGD cofactors.4,11,13 The electron-transferring ß-subunit (FdnH/FdoH) has four [4Fe-4S] clusters, while the γ-subunit (FdnI/FdoI) is an integral membrane protein that anchors the other two subunits to the periplasmic site of the membrane. The γ-subunit possesses two heme b groups and a menaquinone binding site.4 The α- and ß-subunits of Fdh-N and Fdh-O are translocated as cofactor-containing heterodimers (FdnGH, FdoGH) across the cytoplasmic membrane via the twin-arginine protein transport (Tat) pathway, a transport system dedicated to the Received: May 16, 2014 Published: September 24, 2014 5524

dx.doi.org/10.1021/pr5004906 | J. Proteome Res. 2014, 13, 5524−5535

Journal of Proteome Research

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transport of fully folded proteins.18,19 Transport of Tat substrates is characteristically mediated by a distinct N-terminal signal sequence comprising a polar N-terminal “n-region” of variable length, followed by a relatively hydrophobic “h-region” of 15−25 amino acids. Both motifs flank a distinct consensus sequence SRRxFLK with the name-giving arginine pair.20,21 Often a positively charged “c-region” within the “h-region” serves as recognition motif for proteolytic cleavage of the signal peptide during substrate transport.20 In the case of the heterooligomeric substrates FdnGH and FdoGH, only the catalytic subunits (FdnG and FdoG) carry a single signal peptide, which mediates translocation into the periplasm, while the ß-subunits (FdnH and FdoH) are believed to be coexported. Such multisubunit molybdoenzymes require the assistance of biosynthetic accessory proteins that ensure the correct insertion of prosthetic groups and coordinate folding events within the cytoplasm;22 however, little is known about whether further interaction partners of the αβ heterodimers of Fdh-N and FdhO support insertion of, or provide scaffolding functions for, these multisubunit enzymes once they enter the periplasm. To address this question, we performed chemical crosslinking in combination with mass spectrometry. During the past 15 years, the chemical cross-linking/MS approach has matured into an alternative technique for analyzing 3D-structures of protein assemblies up to megadalton size.23−31 The strength of the cross-linking/MS approach is that experiments can be conducted with purified proteins under native-like conditions with respect to pH and ionic strength. The ultimate goal is to conduct cross-linking experiments in living cells,32 but so far mainly highly abundant proteins have been identified when such cross-linking reactions have been conducted using living cells. While for most structural proteomics applications homobifunctional amine-reactive cross-linkers are applied, heterobifunctional amine/photo-reactive linkers exhibit distinct advantages.33,34 As such, the bait protein can be activated by reaction with the amine-reactive site of the cross-linker before potential interaction partners are added, and the formation of a covalent bond is induced by UV irradiation via the photoreactive site of the linker. Here we present the identification of interaction partners of Fdh-N and Fdh-O from a subproteome enriched in periplasmic proteins using the heterobifunctional amine/photo-reactive cross-linker SDAD followed by MS analysis of the cross-linked products. For four selected periplasmic Fdh-binding partners (Skp, ErfK, OppA, and TolB), we also mapped the interfaces within the Fdh/protein complexes. These studies allow us to derive structural and functional information on the mechanisms underlying the interactions between the multisubunit proteins, Fdh-N and Fdh-O, and cell envelope binding proteins that potentially play a role in their stabilization and functionality when in the periplasm.

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Purification of Formate Dehydrogenases

Because of the high sequence similarity between Fdh-O and Fdh-N, both proteins copurified during the isolation procedure. 50 g wet-weight of cells was resuspended in 150 mL of 50 mM MOPS, 1 μM DNase I, 2 mM PMSF, pH 7.5 and disrupted by three passages through a French Press. Subsequent steps were carried out at 0−4 °C unless stated otherwise. The lysate was centrifuged at 5000 rpm for 30 min. Subsequently, the supernatant was centrifuged at 25 000g for 30 min. The cellfree supernatant was then centrifuged at 40 000 rpm for 2 h using a Beckman Coulter ultracentrifuge (Beckman Coulter, Optima L Series; Type 45 Ti Rotor). The membrane fraction was resuspended in 50 mM MOPS, 0.5% (w/v) DOC, pH 7.5, and the protein concentration was adjusted to 5 mg/mL. After 10 min of incubation, solid ammonium sulfate was slowly added to a concentration of 30%. The suspension was stirred for 1 h, and the precipitate was removed by centrifugation at 28 000g for 30 min. The supernatant was used to generate a 50% ammonium sulfate protein fraction. After centrifugation at 28 000g for 30 min again, the precipitate from the 50% ammonium sulfate fractionation was recovered in 10 mL of 50 mM MOPS, 1 M ammonium sulfate, pH 7.5. Protein purification was performed using an Ä KTA FPLC system (GE Healthcare). The mixture was applied to a butyl sepharose column (4.9 mL; GE Healthcare) pre-equilibrated with at least 10 column volumes of equilibration buffer (50 mM MOPS, 1 M ammonium sulfate, pH 7.5). A gradient from 1 to 0 M ammonium sulfate (in 50 mM MOPS, pH 7.5) was applied over 10 column volumes. Proteins were eluted with Milli-Q-water containing 0.5% (w/v) Triton X-100. All fractions showing Fdh activity were pooled and concentrated to a volume of 2 mL using an Amicon filter (10-kDa cutoff, Millipore). Simultaneously, buffer was exchanged to 50 mM MOPS, 1 M NaCl, 0.5% (w/v) Triton X-100, pH 7.5. The concentrated protein solution was applied to a Sephacryl S400 size-exclusion column (320 mL; GE Healthcare) pre-equilibrated with two column volumes of buffer (50 mM MOPS, 1 M NaCl, 0.5% (w/v) Triton X-100, pH 7.5), and the column was developed using the same buffer. All fractions showing Fdh activity were pooled, the solution was concentrated to a volume of 2 mL, and the buffer was exchanged to 50 mM MOPS, pH 7.5. This sample was applied to an anion exchange column (MonoQ 5/50 GL, Amersham Biosciences) equilibrated with 50 mM MOPS, pH 7.5, 0.1% (w/v) Triton X-100). The column was washed with equilibration buffer before proteins were eluted with a linear gradient from 0 to 100% using 50 mM MOPS, 1 M NaCl, 0.1% (w/v) Triton X-100, pH 7.5. Fdh-containing fractions were concentrated, rapidly frozen in liquid nitrogen, and stored at −20 °C until use. Enzyme Assay

Fdh-containing fractions were identified by the formatedependent reduction of 2,4-dichlorophenolindophenol (DCPIP) in the presence of phenazine methosulfate at 37 °C, as described.13 The reaction mixture (1 mL) contained 50 mM MOPS, 80 mM sodium formate, 260 μM PMS, 120 μM DCPIP, pH 7.5. The reduction of DCPIP was initiated by adding 5−50 μL of protein solution.

EXPERIMENTAL PROCEDURES

Growth Conditions

E. coli wild-type strain MC4100 was cultivated in 3 × 5 L batch cultures at 37 °C. Cells were grown anaerobically in TGYEP medium,35 supplemented with 100 mM potassium nitrate, 2 μM sodium selenite, and 2 μM sodium molybdate. At the end of exponential growth, cells were rapidly cooled to 0−4 °C and harvested by centrifugation. Finally, cells were shock-frozen in liquid nitrogen and stored at −20 °C.

SDS-PAGE and In-Gel Digestion

SDS-PAGE (12% acrylamide) under nonreducing conditions was used to visualize Fdh complexes. After staining with Coomassie Brilliant Blue solution, gel slices containing cross5525



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solution of 8 M urea, 400 mM ammonium bicarbonate was added, and the samples were sonicated for 20 min. After dilution with 170 μL of water and further sonication for 5 min, 0.1 μg trypsin or a mixture of trypsin/GluC was added, and incubation was performed overnight at 37 °C.

linked complexes were excised and in-gel digested with trypsin (Promega) following a standard protocol.36 Preparation of the Periplasmically-Enriched Subproteome

At the end of exponential growth, cells from 4 × 5-L cultures were cooled to 0−4 °C and pelleted by centrifugation at 5000 rpm for 30 min. Harvested cells were carefully washed three times with 800 mL of 50 mM HEPES (pH 8.0) each. The final pellet was recovered in 140 mL of 50 mM HEPES (pH 8.0) and 20% sucrose. Afterward, 70 mL of 100 mM EDTA pH 8.0 (1 mg lysozyme/mL) was added and incubated for 5 min on ice. Generated spheroplasts were pelleted by centrifugation at 13 000 rpm for 20 min. Finally, the supernatant composed of cell envelope and periplasmic proteins was decanted, shockfrozen in liquid nitrogen, and stored at −20 °C until use.

Liquid Chromatography/Mass Spectrometry

In-gel-digested peptides were analyzed immediately by LC−MS on an Ultimate nano-HPLC system (Dionex/Thermo Fisher Scientific) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ionization (ESI) source (Proxeon). The 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% (w/v) 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% (v/v) acetonitrile, 0.1% (v/v) formic acid). Peptides were separated with linear gradients from 0 to 40% B (80% (v/v) acetonitrile, 0.08% (v/v) formic acid) over 90 or 180 min. The column was kept at 30 °C and the flow-rate was set to 300 nL/min. Online MS data were collected in data-dependent MS/MS mode during the complete gradient elution: Each high-resolution full scan (m/z 300 to 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 2.3 Th). Dynamic exclusion (repeat count was 2, exclusion duration 120 s) was enabled to allow detection of less abundant ions. Data analysis was performed using the Proteome Discoverer 1.3 (Thermo Fisher Scientific), and MS/MS data of precursor ions (m/z 500−5000) were searched against the SwissProt Database (version 05/2013, taxonomy E. coli, 22 937 entries) using Mascot (version 2.2, Matrixscience). Mass accuracy was set to 10 ppm and 0.8 Da for precursor and fragment ions, respectively. Carbamidomethylation of cysteine and oxidation of methionine were set as potential modifications, and up to three missed cleavages of trypsin were allowed.

Subfractionation of the Periplasmically-Enriched Subproteome

To reduce the complexity of the cell envelope subproteome, we applied a sample (190 mg protein) to a size-exclusion chromatography (SEC) Superdex S75 16/600 GL column (GE Healthcare) pre-equilibrated with two column volumes of 50 mM HEPES (pH 7.5), 250 mM NaCl. The flow-rate was set to 0.75 mL/min. Ten fractions, with a fractionation volume of ca. 3 mL each, were collected. SDAD Cross-Linking

Fdh-containing fractions were diluted with 50 mM HEPES (pH 8.0), 100 mM NaCl, and 0.007% DDM to give a final protein concentration of 35.7 μM. A 144 mM solution of SDAD (succinimidyl 2-([4,4′-azipentanamido]ethyl)-1,3′-dithiopropionate; Thermo Fisher Scientific) in DMSO was added in 140fold molar excess of cross-linker over protein. Fdh’s were modified via the amine-reactive site of SDAD by incubation for 2 h at 0 °C in the dark. Removal of nonreacted cross-linker was achieved using Amicon Ultra filtration units (0.5 mL, 10 kDa, Millipore). Afterward, the sample was recovered in 50 mM HEPES buffer (pH 8.0) containing 100 mM NaCl, 0.007% DDM. A subfraction of cell envelope proteins (MWaverage of 45 kDa) was recovered in 50 mM HEPES (pH 8.0), 100 mM NaCl, and 0.007% DDM to give a final protein concentration of 178.5 μM. SDAD-modified Fdh’s and cell envelope subfraction were mixed at a 1:1 ratio (v/v) and reconcentrated to 35.7 μM Fdh-N and 178.5 μM cell envelope proteins. Induction of photo-cross-linking between Fdh’s and its binding partners was implemented by irradiation with UV-A light (maximum at 365 nm, 8000 mJ/cm2).

Identification of Cross-linked Products

Assignment of cross-linked products was performed with GPMAW (General Protein Mass Analysis for Windows) software, version 8.1 (Lighthouse Data, www.gpmaw.com) and the in-house software StavroX.37 StavroX was used for automatic comparison of MS and MS/MS data from Mascot generic format (mgf) files. Potential cross-links were manually evaluated. A maximum mass deviation of 10 ppm between theoretical and experimental mass and a signal-to-noise ratio (S/N) ≥ 2 was allowed. Lys, Ser, Thr, Tyr, and N-termini were considered as potential reaction sites for the amine-reactive site of the cross-linker SDAD.38 All amino acids were taken into account as potential cross-linking sites for the photoreactive diazirine group of SDAD. Because the carbene intermediate is highly reactive, diazirines will react with a large number of amino acids.39 Oxidation of Met was considered to be a potential modification, in addition to three missed cleavage sites for each amino acid Lys, Arg, Glu, and Asp.

Enrichment of Cross-Linked Products

For an enrichment of cross-linked protein complexes, 100 μL of cross-linked sample (protein concentration 110 μM) was subjected to SEC using a Superdex-200 16/300 GL column (GE Healthcare) and pre-equilibrated with two column volumes of 50 mM HEPES (pH 8.0), 100 mM NaCl, 0.007% DDM. In-Solution Digest of Protein Complexes

SEC fractions eluting between 7 and 10, 10−13, 13−15, and 15−18 mL were pooled and concentrated to a final volume of 100 μL each using Amicon Ultra filtration units (0.5 mL, 10 kDa, Millipore). Proteins were precipitated by the addition of 500 μL of acetone and overnight incubation at −20 °C. The samples were centrifuged at 16 000g for 1 h, and the supernatants were discarded. The precipitates were dried using a vacuum concentrator (miVac). Then, 25 μL of a 5526



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Figure 1. Experimental workflow established for binding partner identification of native multisubunit protein complexes. Left column: A subproteome containing cell envelope proteins is prepared, which is subjected to SEC to further reduce its complexity. Middle column: Purified Fdh’s are labeled with the amine-reactive site of the cross-linker SDAD before cell envelope proteins are added. Cross-linking between Fdh’s and binding partners from the cell envelope preparation is induced by UV-A irradiation. An enrichment of cross-linked complexes is performed by SEC. Right column: Fractions enriched in cross-linked complexes are enzymatically digested, and Fdh binding partners are identified by LC−MS/MS analysis based on peptide spectral matches in the cross-linking reaction mixture and a non-cross-linked control sample. The exact cross-linked sites between Fdh’s and binding proteins are identified by analyzing MS/MS data with the in-house StavroX software.

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RESULTS

heterobifunctional cross-linker SDAD, and the subproteome enriched for periplasmic proteins was added and cross-linked to the SDAD-modified Fdh’s by UV irradiation. Cross-linked protein complexes can be enriched via the affinity tag of the bait protein. However, a native purification of the bait protein possesses the inherent advantage that cellular processes are not disturbed by overexpression, and, moreover, potential protein interactions are not influenced by the tag. Here we succeeded in purifying Fdh-N and Fdh-O under native conditions, which comprised mainly FdnGH and FdoGH heterodimers without the membrane-anchor I subunit, followed by an SEC-based enrichment of cross-linked Fdh’s complexes (Figure 1). Elution fractions were collected, and Fdh/protein complex-containing fractions were applied to in-solution digestion. Afterward, the resulting peptide mixtures were subjected to nano-HPLC/ nano-ESI-LTQ-Orbitrap MS/MS analysis, and potential Fdh interaction partners were screened by spectral counting of MS/ MS spectra from the cross-linked sample and non-cross-linked control. For the four selected binding partners, Skp, OppA, ErfK, and TolB, we also mapped the exact interaction regions in the respective Fdh/protein complexes.

Analytical Strategy for the Identification of Fdh Interaction Partners

The aim of this study was to identify periplasmic binding partners of the two respiratory Fdh’s in E. coli (Figure 1), which might have important roles in stabilizing or “scaffolding” these large oxidoreductases in the periplasm. Chemical cross-linking combined with a mass spectrometric analysis of the generated cross-links is a powerful method for capturing less abundant and transient interaction partners by the introduction of covalent bonds. In vitro cross-linking experiments often use purified proteins that are incubated with cell lysates to identify potential binding proteins.29,32 However, the presence of highly abundant proteins within complex protein mixtures might lead to false-positives, and this hampers the detection of lowabundance cross-linked products. Therefore, we decided to use a subproteome enriched from the periplasmic fraction of E. coli to search for potential binding partners of Fdh’s. The subproteome fraction was prepared by initially isolating the periplasmic and outer membrane fractions of the cell envelope, and we further reduced its complexity by SEC (see Experimental Procedures). This resulted in a subproteome that was enriched in periplasmic proteins (see below). The two native E. coli Fdh isoenzymes, Fdh-N and Fdh-O, were purified to serve as bait proteins for potential binding partners. In this context, it has to be mentioned that we identified mainly peptides from subunits FdnGH and FdoGH but only very few peptides from subunit FdnI/FdoI after the purification procedure. Fdh-N and Fdh-O were then modified by the

Isolation of Cell Envelope Proteins Containing Potential Fdh Binding Partners

The E. coli subproteome enriched in periplasmic proteins and which contained potential binding partners of Fdh-N and FdhO was prepared using the osmotic shock lysozyme-EDTA method (see Experimental Procedures). The cell envelope subfraction was qualitatively assessed for the presence of cytoplasmic contaminants. For this, proteins were separated by 5527



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Table 2. Classification of Proteins Identified in E. coli Strain MC4100 Envelope Extract Prepared by Osmotic Shock, Lysozyme EDTA Treatment According to Their Subcellular Localization, and Abundance Determined by MS/MS

SDS-PAGE (Figure 2, Lane L), subjected to in-gel digestion, and analyzed by nano-HPLC/nano-ESI-LTQ-Orbitrap MS/

subcellular localization

total no. of proteins

peptide spectral counts

peptide ratio [%]

periplasmic outer membrane inner membrane cytoplasmic uncharacterized total

129 12 20 158 17 336

5381 122 280 1721 266 7776

69.3 1.6 3.6 22.1 3.4 100

report on an optimized TSE (tris-sucrose solution supplemented with EDTA) preparation of cell envelope proteins.41 Notably, 74.4% (5783 peptides) of all peptides identified originated from cell envelope proteins (periplasmic, inner membrane, and outer membrane proteins, Figure 3). Only 17 uncharacterized proteins (266 peptides) could not be assigned to a specific cellular compartment due to a lack of information. The high-abundance proteins identified are shown in Table 1. Among the 100 most abundant proteins, only 19 were localized in the cytoplasm. The remaining proteins were from either the periplasmic, the inner membrane, or the outer membrane fractions. These data illustrate the efficient enrichment of the cell envelope, which contains potential Fdh interaction partners, together with some cytoplasmic contaminants. Prior to chemical cross-linking, the cell envelope subfraction was further reduced in complexity by SEC (Figure 1). Ten subfractions of lower complexity were generated, and the quality of the chromatographic separation was evaluated by SDS-PAGE analysis of the fractions (Figure 2). We decided to combine elution fractions 5 and 6 because they optimally

Figure 2. SDS-PAGE analysis of periplasmic complexity reduction by SEC (Superdex S75 16/600 GL column). The cell envelope subproteome was prepared from MC4100 cells cultivated in TGYEP medium. 10% (w/v) resolving gel; M: molecular weight marker; L: sample applied to the column; 1−10: elution fractions. Proteins in lane L were subjected to in-gel digestion and identified by nano-HPLC/ nano-ESI-LTQ-Orbitrap MS and MS/MS analysis (Table 1 and Table S1, Supporting Information).

MS. For a rough estimation of protein abundances, we used spectral counting for each protein identified (Table 1 and Table S1, Supporting Information). The spectral counts reflect the number of MS/MS spectra measured for peptides of a specific protein.40 Mass spectrometric analysis of the cell envelope subfraction led to the identification of 129 periplasmic proteins, 12 outer membrane proteins, 20 inner membrane proteins, 158 cytoplasmic proteins, and 17 uncharacterized proteins (Table 2 and Table S1, Supporting Information). The number of spectral counts originating from cytoplasmic contaminants was 22.1% (1721 peptides), which is comparable to a previous Table 1. Subproteome Analysis of Cell Envelope Preparationa UniProtKB entry

protein

gene name

subcellular location

peptide spectral counts

sequence coverage [%]

P37329 P0AFH9 P23843 P24183 P33363 P0AFM3 P45523 P23865 P0C0V1 P16700 P09373 P0AEN0 P07024 P0ABZ8 A7ZKF2 P05458 A1A7M1 A7ZSL4 P39099 C4ZU48 A7ZJC2

molybdate-binding periplasmic protein osmotically inducible protein y periplasmic oligopeptide-binding protein formate dehydrogenase, major subunit periplasmic beta-glucosidase glycine betaine-binding periplasmic protein FKBP-type peptidyl-prolyl cis−trans isomerase tail-specific protease periplasmic serine endoprotease thiosulfate-binding protein formate acetyltransferase cystine-binding periplasmic protein protein UshA chaperone SurA glucans biosynthesis protein protease 3 outer membrane protein assembly factor elongation factor Tu 1 periplasmic pH-dependent serine endoprotease periplasmic nitrate reductase protein TolB

modA osmY oppA fdnG bglX proX f kpA Prc degP cysP pf lB f liY ushA surA mdoG ptrA bamA tuf1 degQ napA tolB

periplasm periplasm periplasm periplasm periplasm periplasm periplasm periplasm periplasm periplasm cytosol periplasm periplasm periplasm periplasm periplasm periplasm cytosol periplasm periplasm periplasm

357 267 249 166 149 144 141 127 122 119 110 109 107 106 95 89 86 82 82 80 80

87.1 70.15 74.59 61.48 66.93 71.52 51.48 47.95 60.34 78.70 54.61 77.44 59.27 56.31 63.21 51.04 40.99 80.46 58.46 46.50 62.79

a

Periplasmic and cell envelope proteins separated by SDS-PAGE (Figure 2, lane L) were subjected to in-gel digestion for subsequent nano-HPLC/ nano-ESI-LTQ-Orbitrap MS/MS analysis of peptides. The proteins with the highest numbers of peptide spectral counts are shown; UniProtKB entry numbers are given. 5528



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between Fdh’s and their cell envelope binding partners and reduces the risk of them being masked by highly abundant noncross-linked peptides during LC−MS analysis. Identification of Interaction Partners

In-solution digestion of the SEC-enriched Fdh/protein complexes was performed, and nano-HPLC/nano-ESI-LTQOrbitrap MS/MS served to analyze the generated cross-linked peptides. To gain insight into the binding partners of Fdh’s, we performed a comparative protein analysis of cross-linked sample and control (Table 3 and Table S2, Supporting Information). Cross-linked Fdh interaction partners were tracked by higher numbers of spectral counts for corresponding proteins in the cross-linked sample compared to the control. The majority of identified proteins exhibited consistent values or only slight variations in spectral counts between the crosslinked sample and the control and as such serve as useful internal standards. In contrast, proteins, such as the inner membrane proteins small-conductance mechanosensitive channel, the α-subunit of respiratory nitrate reductase (Nar) 1, and the periplasmic chaperone Skp, displayed more pronounced differences in spectral count ratios (Table 3). The soluble periplasmic proteins ErfK (a probable L,D-transpeptidase) and the oligopeptide-binding protein OppA also showed major differences in spectral counts between cross-linked and control samples. We therefore chose these periplasmic and inner membrane proteins that were enriched in the cross-linked sample for a more detailed inspection of the interfaces in the complexes with Fdh’s. In addition, we decided to investigate the interface regions in the Fdh/TolB complex because in complementary conducted experiments TolB was found to be enriched in a fraction within a smaller elution window (retention volumes 9 to 10 min) during SEC in the crosslinked mixture versus the control sample (data not shown). The Tol/Pal proteins of E. coli are located in the periplasm facing either the inner or the outer membrane and are responsible for maintaining the membrane integrity.42−45 The Tol/Pal complex connects the inner and outer membrane leaflets and extends through the periplasm and TolB associates with this nanomachine, interacting also with the peptidogly-

Figure 3. Classification of peptides identified in E. coli strain MC410 envelope extract prepared by osmotic shock, lysozyme EDTA treatment according to their subcellular localization, and abundance determined by mass spectrometry. Values are given in percentage.

represent E. coli periplasmic proteins ranging between 10 and 70 kDa. Subsequently, combined fractions 5 and 6 (Figure 2, lanes 5 and 6) were subjected to chemical cross-linking with the purified Fdh’s. The SDAD-cross-linking reaction was monitored by SDS-PAGE, revealing distinct signals that are indicative of the formation of cross-linked Fdh complexes (Supporting Information, Figure S1). Enrichment of Cross-Linked Products

For an enrichment of cross-linked Fdh’s complexes, the SDADcross-linking reaction mixture was subjected to SEC (Figures 1 and 4). The chromatograms of the SDAD reaction mixture and non-cross-linked control were superimposed for detection of differences in their elution profiles (Figure 4). Significantly higher absorption values were measured at 280 nm within retention volumes between 8 and 13 mL for the cross-linked sample compared to the control. This indicates the emergence of molecules with high molecular weights, that is, Fdh/protein complexes, upon cross-linking. The major peak with a retention volume of 14 to 18 mL corresponded to the periplasm-enriched protein fraction that was not cross-linked to Fdh’s. The SEC elution profile gives first hints on a separation of Fdh/protein complexes and non-cross-linked proteins. The successful enrichment of cross-linked Fdh/protein complexes is the basis for a successful detection of intermolecular cross-links

Figure 4. Enrichment of SDAD-cross-linking products via SEC (Superdex S200 16/300 GL column). Chromatogram of SDAD-reaction mixture (solid line) and non-cross-linked sample (control, dashed line) are shown. UV absorption at 280 nm of both elution profiles was normalized. 5529



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Table 3. Comparative Analysis of Peptides Identified in Cross-Linked and Non-Cross-Linked (control) Sample after SEC Using a Superdex S200 16/300 GL Columna peptide spectral counts UniProtKB entry P24183 P32176 P0C0S2 P0A9M8 A7ZTU4 P09152 A7ZHA4 P0AAJ3 P23843 P0AEU9 P63388 P39176 A1AJ51 P0A9Q8

protein name

cross-linked sample

control sample

spectral counts ratio (cross-linked/control sample)

formate dehydrogenase-N, major subunit formate dehydrogenase-O, major subunit small-conductance mechanosensitive channel phosphate acetyltransferase ATP synthase subunit beta respiratory nitrate reductase 1 alpha chain chaperone protein DnaK formate dehydrogenase N, iron−sulfur subunit periplasmic oligopeptide-binding protein chaperone protein skp probable phospholipid import ATP-binding protein probable L,D-transpeptidase ErfK 60 kDa chaperonin 1 aldehyde-alcohol dehydrogenase

158 77 75 54 53 45 35 34 31 31 28 27 26 23

160 77 23 57 58 10 32 34 19 0 24 14 23 28

0.98 1 3.26 0.94 0.91 4.5 1.09 1 1.63 1.16 1.92 1.13 0.82

a

Elution fractions within retention volumes of 7−13 mL (Figure 4) were pooled, digested in solution, and subjected to nano-HPLC/nano-ESI-LTQOrbitrap MS/MS analysis. UniprotKB entry numbers are given.

Table 4. Identified Intermolecular Cross-Linksa

a

Cross-linked amino acids are shown in red.

can.46 As well as being important for stabilizing the membrane, the Tol machinery is a conduit for the uptake of colicins.47 It is conceivable, therefore, that the Tol machinery might have a further function in associating with and stabilizing large periplasmic respiratory enzymes, such as Fdh-N and Fdh-O, and hence our interest in investigating the interaction of Fdh-N with TolB.

latter two proteins have the potential to interact with Fdh’s only via their membrane-spanning regions, whereas their cytoplasmic regions are believed to be spatially distant from Fdh’s in the cell and thus cannot be cross-linked. The StavroX scores of the cross-links identified in the Fdh/protein complexes are presented in Table S3, Supporting Information. Among the cross-links identified, four distinct cross-links were between FdnH and Skp, three between FdnG and OppA, and one between FdnG and ErfK (Table 4 and Figure S2, Supporting Information). We also found three cross-links between FdnH and TolB and two cross-links between FdnG and TolB. In this context, it has to be noted that G- and Hsubunits of Fdh-N and Fdh-O share amino acid sequence identities of ca. 76 and 75%, respectively. In 3 out of 13 crosslinked products, this led to an uncertainty whether Fdh-N or

Mapping Interfaces of Fdh/Cell Envelope and Periplasm Protein Complexes

Thirteen intermolecular cross-links were unambiguously identified between Fdh’s and their potential cell envelope and periplasm protein partners, Skp, OppA, ErfK, and TolB (Table 4, Figure 5, and Figure S2, Supporting Information), while for the small-conductance mechanosensitive channel and Nar no cross-links were identified. This is not surprising because the 5530



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Figure 5. Nano-HPLC/nano-ESI-LTQ-Orbitrap MS/MS analysis of intermolecular cross-links of (A) FdnH with Skp and (B) FdnG with OppA. Precursor ions were selected, fragmented, and analyzed in the linear ion trap. FdnG and FdnH peptide fragment ions are colored in red, whereas Skp and OppA fragment ions are colored in blue. (A) Fragment ion mass spectrum of the 2+ charged precursor ion at m/z 1260.684. The cross-linked product comprises amino acids 91−97 of FdnH (peptide shown in red) and amino acids 81−91 of Skp (peptide shown in blue), in which K97 of FdnH is connected to T81 or K82 of Skp. (B) Fragment ion mass spectrum of the 3+ charged precursor ion at m/z 877.082. The cross-linked product comprises amino acids 67−79 of FdnG (peptide shown in red) and amino acids 164−171 of OppA (peptide shown in blue), in which Y76 of FdnG is connected to D167 of OppA.

Fdh-O in fact formed the cross-link (Table 4). Except for TolB, all cross-links with Skp, OppA, and ErfK were found to either the FdnG or FdnH subunits of Fdh’s. MS/MS data of the intermolecular cross-links are exemplarily shown for the interaction products of FdnH with Skp (Figure 5A) and FdnG with OppA (Figure 5B). The cross-linked product between FdnH and Skp comprises amino acids Leu-91 to Lys-97 (FdnH) and Thr-81 to Arg-91 (Skp), in which Lys96 (FdnH) is connected to Thr-81 or Lys-82 (Skp). The crosslinked product between FdnG and OppA comprises amino acids Gly-67 to Glu-79 (FdnG) and Ala-164 to Glu-171 (OppA), in which Tyr-76 (FdnG) is connected to Asp-167 (OppA). When conducting cross-linking experiments, it is of utmost importance to evaluate the quality of the scoring of the crosslinking software and to reduce the identification of falsepositive cross-links to the greatest extent possible. Therefore,

we checked for intramolecular cross-links within FdnG and FdnH as well as for intermolecular cross-links between FdnG and FdnH as for both Fdh-N subunits 3D-structural information is available (PDB entry 1KQF). Indeed, almost all of the high-scoring cross-links were found to exhibit Cα−Cα distances up to a maximum of ca. 32 Å, which is in agreement with the spacer length (13.5 Å) of the cross-linker SDAD used in this study.28 Only two cross-links were found to bridge slightly longer distances within FdnG or between FdnG and FdnH (∼35 and 36 Å), which can explained by the inherent flexibility of the protein. The low-scoring cross-links within FdnG and FdnH exhibit much longer Cα−Cα distances (Tables S4 A−E and Figure S3, Supporting Information). We are therefore confident in the scoring algorithm StavroX uses and the correct identification of our cross-links. A summary of the recorded MS and MS/MS data together with the number of 5531



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cross-links passing the StavroX significance filter is presented in Table S5 (Supporting Information).

broad, comprising more than 30 different proteins,53 and the chaperone has been proposed to have a function in preventing protein aggregation rather than folding.54 Skp binds to outermembrane protein substrates with dissociation constants in the low nanomolar range.55 Skp exhibits a domain selective activity whereby for a bidomain substrate the transmembrane domain is bound by the chaperone, whereas the soluble domain protrudes from this complex and adopts its native fold.56 Conformations and backbone dynamics of the Skp-OMP complex have been presented.57 In vitro experiments reveal that Skp helps to fold soluble proteins by inhibiting aggregation.58 However, little is known about the mechanisms, by which Skp enhances or stabilizes folding of soluble or inner membrane proteins, in particular, of Tat substrates. From the cross-links identified, it is clear that all lysine residues in FdnH that are cross-linked with Skp are aligned (Figure 6A). Moreover, in Skp, the cross-linked amino acids are also aligned. Apparently, the Skp binding interface is located in the central part of FdhH. Interestingly, no cross-link was found in the protruding α-helix representing the membrane anchor of FdnH, indicating the validity of our cross-linking strategy. Because the membrane anchor is usually inaccessible to binding partners, no cross-linked products are found in this region.

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DISCUSSION A number of cell envelope and periplasm protein binding partners of Fdh’s were identified after subproteome fractionation using a cross-linking/MS approach. The cross-linking sites of four potential Fdn binding partners, namely, Skp, OppA, ErfK, and TolB, were studied in detail to map their interface regions with Fdh’s. FdnH/Skp Interface

The 17-kDa protein Skp is an ATP-independent general periplasmic chaperone present in many Gram-negative bacteria that is involved in the folding and the insertion of proteins into the outer membrane.48−50 The crystal structure of Skp reveals a trimer with a “jellyfish” architecture where a cavity is formed by long tentacle-like helical protrusions emanating from a body domain (Figure 6).51,52 The biological substrate range of Skp is

FdnG/OppA Interface

OppABCDF is an ATP-dependent oligopeptide transporter that is a member of the ATP-binding cassette (ABC) superfamily of transporters.59 The system has been found to act in oligopeptide uptake as well as in recycling of cell-wall peptides.60,61 OppA is the periplasmic substrate-binding component that binds to oligopeptides with dissociation constants in the low micromolar range62 and is also a substrate of Skp.54 OppA has been crystallized, and its structure has been resolved to 2.3 A resolution showing OppA to be a bilobed, principally ß-stranded, three-domain protein.63 The residues in FdnG that were found to be cross-linked to OppA comprise two serines, and one tyrosine residue as hydroxyl groups can also react with the amine-reactive site of the cross-linker SDAD (Figure 6B).38 As was observed for the FdnH/Skp complex, the cross-linked amino acids are facing in one direction. In OppA, the cross-linked amino acids are spatially separated from each other; however, they are also aligned with one another. The interface between FdnG and OppA can be envisaged from the cross-linked amino acids as spanning one side of the OppA protein. FdnG/ErfK Interface

ErfK is an L,D-transpeptidase responsible for the removal of the D-alanine residue of peptidoglycan tetrapeptide stems and the attachment of the lysine residue of Braun lipoprotein to the meso-diaminopimelyl (DAP) residue of the resulting tripeptide. This activity results in a covalent attachment of peptidoglycans to the outer membrane and assures cell-wall integrity. Loss of all Ldt enzymes is detrimental to growth when cells are starved for DAP, indicating a key role for ErfK in dealing with peptidoglycan stress.64 For ErfK, only one cross-link was found to the FdnG subunit (Table 4), making it difficult to draw more detailed conclusions on the interface region in the ErfK/FdnG complex. Because the large Fdh enzyme complexes likely contact the peptidoglycan layers, this might provide a biological rationale for this specific interaction. Moreover, the fact that this single cross-linking site that was found in FdnG (Lys-138) is different from the

Figure 6. Identification of intermolecular cross-links of FdnH with Skp and FdnG with OppA. The overall structures of the FdnG (blue), FdnH (blue), Skp trimer (green), and OppA (green) are represented as ribbon diagrams (PDB entries 1SG2 and 1KQF). The identified cross-linking sites are represented as red sticks, respectively. (A) SkpFdnH cross-links. Four cross-links were found between Skp (T118 or R119, R59, M74, and T81 or K82) and FdnH (K202, K204, K110, and K96). Identified cross-linking reaction sites of the Skp trimer are shown for subunit A. (B) OppA-FdnG cross-links. Three cross-links were found between OppA (A381 or D382, Y121, and D167) and FdnG (S125, S183, and Y76). 5532



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interaction sites of Skp, OppA, and TolB suggests that the ErfK/FdnG interface differs from that of OppA and TolB.

ASSOCIATED CONTENT

S Supporting Information *

FdnG/TolB Interface

Subproteome analysis of cell envelope preparation, comparative analysis of peptides identified in cross-linked and non-crosslinked control sample after SEC, qualitative assessment of FdhSDAD-labeling and chemical cross-linking, LC−MS/MS data of all Fdh/protein cross-linked products identified, and presentation of cross-linked residues in the 3D-structures of FdnGH and TolB. This material is available free of charge via the Internet at http://pubs.acs.org.

The Tol/Pal system of the E. coli cell envelope is composed of six proteins: TolQ, TolR, and TolA form a complex in the inner membrane,42 while TolB, a periplasmic protein, interacts with the peptidoglycan-associated lipoprotein (Pal), which is anchored to the outer membrane and interacts with the Cterminal domain of TolA.65 TolB has been shown to interact in vivo with the major outer membrane lipoprotein and OmpA in a Pal-dependent manner.66 These results provide strong evidence that the Tol/Pal system functions to maintain cell envelope integrity.65 Tol proteins are responsible for maintaining the colicins in an unfolded conformation, aiding in their translocation across the periplasm and targeting them to the cytoplasmic membrane or cytoplasm.67 In contrast with the three aforementioned Fdh-binding partners, where cross-links were identified to only one of the subunits, FdnG or FdnH, TolB was found to interact with both subunits, FdnG and FdnH (Figure S4, Supporting Information). The residues in Fdh interacting with TolB are not identical to those found for the other three periplasmic proteins described above. Again, no cross-link was identified in the αhelix representing the membrane anchor of FdnH, as this is inaccessible for binding. Strikingly, the FdnH subunit binds (Figure S4, Supporting Information, cross-linked amino acids are marked in green) to different regions of TolB compared with the subunit FdnG (Figure S4, Supporting Information, cross-linked amino acids are marked in red), and from the cross-linking pattern, one can deduce the interface regions between Fdn and TolB. These interactions along the length of the Fdh-N enzyme suggest that TolB might provide a stabilizing function for the Fdh. Furthermore, the fact that TolB is peptidoglycan-associated is supported by the identification of ErfK as an interaction partner of FdnG. Conclusively, we were able to identify binding partners of formate dehydrogenases from a periplasmic subproteome. So far, there are no characterized interaction partners for Fdh-O. For FdnG, AceE, FdhE, TufA, and TufB have been reported as binding partners, while for FdnH, there is one known interaction with HolA.68

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.Z. is supported by the DFG-Graduiertenkolleg GRK 1026 “Conformational Transitions in Macromolecular Interactions”. A.S. and R.G.S. acknowledge financial support from the DFG and the region of Sachsen-Anhalt. Christine Piotrowski and Dirk Tänzler are acknowledged for support with protein purification. Isabel Kratochvil is acknowledged for assistance in analyzing the cross-linking datasets.

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ABBREVIATIONS CID, collision-induced dissociation; DAP, diaminopimelic acid; DDM, n-dodecyl-β-D-maltoside; DCPIP, 2,4-dichlorophenolindophenol; DMSO, dimethyl sulfoxide; DOC, sodium deoxycholic acid; ErfK, probableL,D-transpeptidase ErfK; ESI, electrospray ionization; Fdh, formate dehydrogenase; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; HPLC, high-performance liquid chromatography; LTQ, linear ion trap (Thermo Fisher Scientific); MS, mass spectrometry; MS/ MS, tandem mass spectrometry; mgf, Mascot generic format; MOPS, 3-(N-morpholino)propanesulfonic acid; Nar, nitrate reductase; OppA, periplasmic oligopeptide-binding protein; Pal, peptidoglycan-associated lipoprotein; pflB, pyruvate formate-lyase B; PMSF, phenylmethylsulfonylfluoride; SDAD, succinimidyl-2-([4,4′-azipentanamido]ethyl)-1,3′-dithiopropionate; SEC, size-exclusion chromatography; Skp, chaperone protein skp; Tat, twin-arginine protein transport; TolB, protein TolB; TSE, tris-sucrose solution supplemented with EDTA

CONCLUSIONS

Using a subproteome fraction of mainly periplasmic proteins for conducting cross-linking/MS experiments, we were able to identify binding partners of formate dehydrogenases that interact with the proteins’ postcytoplasmic maturation and translocation into the periplasm. We were also able to gain detailed structural information on the interfaces of four selected Fdh complexes. Notably, each interaction partner bound at mutually exclusive sites. The interaction sites of Fdh subunits were mapped for the general periplasmic chaperone Skp, the peptidoglycan-binding proteins ErfK and TolB, as well as the oligopeptide-binding and Skp-interacting OppA. Future studies will aim to clarify how these proteins likely aid in the final docking of the Fdh GH heterodimers with its respective membrane anchoring I subunit as well how they accommodate and organize these large multisubunit enzymes with the peptidoglycan layer in the periplasmic space.

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