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Surface-exposed domains of TatB involved in the structural and functional assembly of the Tat translocase in Escherichia coli

  • Julia Fröbel
    Footnotes
    Affiliations
    Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany
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  • Anne-Sophie Blümmel
    Footnotes
    Affiliations
    Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany
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  • Friedel Drepper
    Affiliations
    Institute of Biology II, Biochemistry–Functional Proteomics, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany

    BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany
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  • Bettina Warscheid
    Affiliations
    Institute of Biology II, Biochemistry–Functional Proteomics, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany

    BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany

    CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany
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  • Matthias Müller
    Correspondence
    To whom correspondence should be addressed: Institute of Biochemistry and Molecular Biology, University of Freiburg, 79104 Freiburg, Germany. Tel.: 49-761-203-5265; Fax: 49-761-203-5274; E-mail: [email protected]
    Affiliations
    Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany
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  • Author Footnotes
    1 Both authors contributed equally to this work.
Open AccessPublished:July 24, 2019DOI:https://doi.org/10.1074/jbc.RA119.009298
      Twin-arginine-dependent translocases transport folded proteins across bacterial, archaeal, and chloroplast membranes. Upon substrate binding, they assemble from hexahelical TatC and single-spanning TatA and TatB membrane proteins. Although structural and functional details of individual Tat subunits have been reported previously, the sequence and dynamics of Tat translocase assembly remain to be determined. Employing the zero-space cross-linker N,N′-dicyclohexylcarbodiimide (DCCD) in combination with LC-MS/MS, we identified as yet unknown intra- and intermolecular contact sites of TatB and TatC. In addition to their established intramembrane binding sites, both proteins were thus found to contact each other through the soluble N terminus of TatC and the interhelical linker region around the conserved glutamyl residue Glu49 of TatB from Escherichia coli. Functional analyses suggested that by interacting with the TatC N terminus, TatB improves the formation of a proficient substrate recognition site of TatC. The Glu49 region of TatB was found also to contact distinct downstream sites of a neighboring TatB molecule and to thereby mediate oligomerization of TatB within the TatBC receptor complex. Finally, we show that global DCCD-mediated cross-linking of TatB and TatC in membrane vesicles or, alternatively, creating covalently linked TatC oligomers prevents TatA from occupying a position close to the TatBC-bound substrate. Collectively, our results are consistent with a circular arrangement of the TatB and TatC units within the TatBC receptor complex and with TatA entering the interior TatBC-binding cavity through lateral gates between TatBC protomers.

      Introduction

      Twin-arginine-dependent translocation denotes the transport of folded proteins across the cytoplasmic membranes of bacteria and archaea and the thylakoidal membrane of plant chloroplasts. Substrate proteins of the twin-arginine translocation (Tat)
      The abbreviations used are: Tat
      twin-arginine translocation
      TM
      transmembrane helix
      APH
      amphipathic helix
      PMF
      proton-motive force
      DCCD
      N,N′-dicyclohexylcarbodiimide
      DCU
      dicyclohexylurea
      NCD-4
      N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl) carbodiimide
      BMOE
      bismaleimidoethane
      NaTT
      sodium tetrathionate
      TmC
      TorA-mCherry
      Bpa
      p-benzoylphenylalanine
      INV
      inside-out inner membrane vesicle(s)
      IAA
      iodoacetamide.
      pathway possess a conserved SRRXFLK sequence motif in their signal sequences. Although the name-giving RR motif is largely indispensable for Tat-specific recognition, other determinants of Tat substrates, such as the hydrophobicity of their signal peptides (
      • Ulfig A.
      • Fröbel J.
      • Lausberg F.
      • Blümmel A.S.
      • Heide A.K.
      • Müller M.
      • Freudl R.
      The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex.
      ,
      • Huang Q.
      • Palmer T.
      Signal peptide hydrophobicity modulates interaction with the twin-arginine translocase.
      ) and sequence characteristics immediately downstream of the signal peptide (
      • Ulfig A.
      • Freudl R.
      The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.
      ), are required for a proficient interaction with the Tat translocase.
      Tat translocases do not seem to preexist in the membrane but rather assemble upon contact with their substrates (
      • Rose P.
      • Fröbel J.
      • Graumann P.L.
      • Müller M.
      Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.
      ,
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ). They assemble from two types of membrane proteins, TatC and one or several members of the TatA protein family. TatC consists of six helices that are tilted and mostly kinked, thus forming the concave structure of a cupped hand or baseball glove (
      • Ramasamy S.
      • Abrol R.
      • Suloway C.J.
      • Clemons Jr., W.M.
      The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.
      ,
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jager F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • et al.
      Structure of the TatC core of the twin-arginine protein transport system.
      ). Two of the six transmembrane helices (TMs) are too short to span the membrane entirely, invoking local membrane perturbations. Similarly short single TMs are encountered at the N termini of all TatA-type proteins. According to NMR structures, the TMs are each followed by a short rigid hinge region and an amphipathic helix (APH) that is predicted to be 50% embedded in the bilayer (
      • Hu Y.
      • Zhao E.
      • Li H.
      • Xia B.
      • Jin C.
      Solution NMR structure of the TatA component of the twin-arginine protein transport system from gram-positive bacterium Bacillus subtilis.
      • Walther T.H.
      • Grage S.L.
      • Roth N.
      • Ulrich A.S.
      Membrane alignment of the pore-forming component TatA(d) of the twin-arginine translocase from Bacillus subtilis resolved by solid-state NMR spectroscopy.
      ,
      • Pettersson P.
      • Ye W.
      • Jakob M.
      • Tannert F.
      • Klösgen R.B.
      • Mäler L.
      Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR.
      ,
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      ). Many Tat translocases involve at least two isoforms of TatA, mostly termed TatA and TatB. TatA and TatB differ in the length of their C-terminal tails distal of the APH. Those tails are largely unstructured but, in the case of TatB, encompass two more helices (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ) that are most likely involved in binding folded substrate proteins (
      • Ulfig A.
      • Freudl R.
      The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.
      ,
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ). A broad range of bacterial Tat translocases involve another homolog of TatA, called TatE (
      • Eimer E.
      • Kao W.C.
      • Fröbel J.
      • Blümmel A.S.
      • Hunte C.
      • Müller M.
      Unanticipated functional diversity among the TatA-type components of the Tat protein translocase.
      ), that is shorter than TatA and TatB. Although TatE shares a high sequence identity with TatA, it represents an autonomous TatA-type subunit (
      • Eimer E.
      • Kao W.C.
      • Fröbel J.
      • Blümmel A.S.
      • Hunte C.
      • Müller M.
      Unanticipated functional diversity among the TatA-type components of the Tat protein translocase.
      ,
      • Eimer E.
      • Fröbel J.
      • Blümmel A.S.
      • Müller M.
      TatE as a regular constituent of bacterial twin-arginine protein translocases.
      ).
      TatA-type proteins bind via overlapping binding sites to the hexahelical TatC (
      • Eimer E.
      • Kao W.C.
      • Fröbel J.
      • Blümmel A.S.
      • Hunte C.
      • Müller M.
      Unanticipated functional diversity among the TatA-type components of the Tat protein translocase.
      ,
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      • Habersetzer J.
      • Moore K.
      • Cherry J.
      • Buchanan G.
      • Stansfeld P.J.
      • Palmer T.
      Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.
      ), leading to the formation of heterooligomeric Tat complexes. The TatB-binding sites of TatC known to date suggest that TatB intercalates between neighboring TatC monomers, thereby giving rise to circular TatBC complexes (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ) with the TMs of TatB associating with each other in the center of the complex (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ). These TatBC arrays are reinforced by distinct TatC–TatC contacts (
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ,
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). The TatBC oligomers provide a deep binding cavity for RR-containing signal sequences (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Huang Q.
      • Alcock F.
      • Kneuper H.
      • Deme J.C.
      • Rollauer S.E.
      • Lea S.M.
      • Berks B.C.
      • Palmer T.
      A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase.
      ,
      • Kreutzenbeck P.
      • Kröger C.
      • Lausberg F.
      • Blaudeck N.
      • Sprenger G.A.
      • Freudl R.
      Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.
      • Lausberg F.
      • Fleckenstein S.
      • Kreutzenbeck P.
      • Fröbel J.
      • Rose P.
      • Müller M.
      • Freudl R.
      Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.
      ) and thus allow their hairpin-like insertion into the plane of the membrane (
      • Ulfig A.
      • Freudl R.
      The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.
      ,
      • Fröbel J.
      • Rose P.
      • Lausberg F.
      • Blümmel A.S.
      • Freudl R.
      • Müller M.
      Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB.
      ,
      • Hamsanathan S.
      • Anthonymuthu T.S.
      • Bageshwar U.K.
      • Musser S.M.
      A hinged signal peptide hairpin enables Tat-dependent protein translocation.
      ). Thus, TatB and TatC jointly function as the substrate receptor of Tat translocases.
      TatC provides a specific recognition site for the RR-consensus motif that is found in the N-terminal part of Tat signal sequences, whereas all TatA-type proteins interact with downstream areas of Tat signal sequences (
      • Ulfig A.
      • Fröbel J.
      • Lausberg F.
      • Blümmel A.S.
      • Heide A.K.
      • Müller M.
      • Freudl R.
      The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex.
      ,
      • Huang Q.
      • Palmer T.
      Signal peptide hydrophobicity modulates interaction with the twin-arginine translocase.
      ,
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jager F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • et al.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Eimer E.
      • Fröbel J.
      • Blümmel A.S.
      • Müller M.
      TatE as a regular constituent of bacterial twin-arginine protein translocases.
      ,
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Taubert J.
      • Brüser T.
      Twin-arginine translocation-arresting protein regions contact TatA and TatB.
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ,
      • Alami M.
      • Lüke I.
      • Deitermann S.
      • Eisner G.
      • Koch H.G.
      • Brunner J.
      • Müller M.
      Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). TatA, which seems to be expressed at superstoichiometric levels compared with the other Tat subunits (
      • Jack R.L.
      • Sargent F.
      • Berks B.C.
      • Sawers G.
      • Palmer T.
      Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth.
      ,
      • Celedon J.M.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      • Hauer R.S.
      • Schlesier R.
      • Heilmann K.
      • Dittmar J.
      • Jakob M.
      • Klösgen R.B.
      Enough is enough: TatA demand during Tat-dependent protein transport.
      ), is unique in that its association with the TatBC complex and with an RR-signal sequence requires the proton-motive force (PMF) at the membrane (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Fröbel J.
      • Rose P.
      • Müller M.
      Early contacts between substrate proteins and TatA translocase component in twin-arginine translocation.
      ). Circumstantial evidence suggested that the N terminus of TatA could destabilize the lipid bilayer (
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      ,
      • Brüser T.
      • Sanders C.
      An alternative model of the twin arginine translocation system.
      ,
      • Hauer R.S.
      • Freudl R.
      • Dittmar J.
      • Jakob M.
      • Klösgen R.B.
      How to achieve Tat transport with alien TatA.
      ), which was recently demonstrated to in fact occur in response to a bound substrate (
      • Hou B.
      • Heidrich E.S.
      • Mehner-Breitfeld D.
      • Brüser T.
      The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding.
      ). This and a PMF-dependent homooligomerization of TatA (
      • Zhang Y.
      • Hu Y.
      • Li H.
      • Jin C.
      Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.
      ,
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      • Dabney-Smith C.
      • Mori H.
      • Cline K.
      Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport.
      ) could be the triggers for the subsequent translocation of folded substrate proteins. Although Tat translocases can transport linear peptide sequences (
      • Richter S.
      • Lindenstrauss U.
      • Lücke C.
      • Bayliss R.
      • Brüser T.
      Functional Tat transport of unstructured, small, hydrophilic proteins.
      ,
      • Cline K.
      • McCaffery M.
      Evidence for a dynamic and transient pathway through the TAT protein transport machinery.
      ), still unknown mechanisms link proficient transport to the folding degree of the substrate proteins (
      • DeLisa M.P.
      • Tullman D.
      • Georgiou G.
      Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway.
      • Panahandeh S.
      • Maurer C.
      • Moser M.
      • DeLisa M.P.
      • Müller M.
      Following the path of a twin-arginine precursor along the TatABC translocase of Escherichia coli.
      ,
      • Richter S.
      • Brüser T.
      Targeting of unfolded PhoA to the TAT translocon of Escherichia coli.
      • Sutherland G.A.
      • Grayson K.J.
      • Adams N.B.P.
      • Mermans D.M.J.
      • Jones A.S.
      • Robertson A.J.
      • Auman D.B.
      • Brindley A.A.
      • Sterpone F.
      • Tuffery P.
      • Derreumaux P.
      • Dutton P.L.
      • Robinson C.
      • Hitchcock A.
      • Hunter C.N.
      Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein.
      ). TatE shares functional properties with both TatA and TatB (
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ,
      • Eimer E.
      • Kao W.C.
      • Fröbel J.
      • Blümmel A.S.
      • Hunte C.
      • Müller M.
      Unanticipated functional diversity among the TatA-type components of the Tat protein translocase.
      ,
      • Eimer E.
      • Fröbel J.
      • Blümmel A.S.
      • Müller M.
      TatE as a regular constituent of bacterial twin-arginine protein translocases.
      ,
      • Baglieri J.
      • Beck D.
      • Vasisht N.
      • Smith C.J.
      • Robinson C.
      Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter.
      ,
      • Sargent F.
      • Bogsch E.G.
      • Stanley N.R.
      • Wexler M.
      • Robinson C.
      • Berks B.C.
      • Palmer T.
      Overlapping functions of components of a bacterial Sec-independent protein export pathway.
      ). Recent data suggest that it is part of the Tat substrate receptor complex and might play a role in the oligomerization of TatA (
      • Eimer E.
      • Kao W.C.
      • Fröbel J.
      • Blümmel A.S.
      • Hunte C.
      • Müller M.
      Unanticipated functional diversity among the TatA-type components of the Tat protein translocase.
      ).
      Although the structures of individual Tat subunits have been solved (see above), current ideas about the possible conformation(s) of a substrate-bound TatABC translocase largely rest on data obtained by biochemical, genetic, and bioinformatics analyses. For a recent model of how the transmembrane parts of TatA, TatB, and TatC might assemble, see to Blümmel et al. (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ). In that report, we introduced N,N′-dicyclohexylcarbodiimide (DCCD) as a new tool to identify contact sites of the Tat subunits. DCCD is a zero-space cross-linker forming isopeptide bonds between carboxyl and amino group-containing side chains of proteins (
      • Valeur E.
      • Bradley M.
      Amide bond formation: beyond the myth of coupling reagents.
      ). By means of a quantitative mass spectrometric analysis of DCCD-treated TatC, we identified distinct intramolecular contact sites of TatC, suggesting that the N-terminal and C-terminal tails of the molecule can tightly pack against the transmembrane core of TatC (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ). Here, we have extended this approach onto the entire TatBC complex based on the premise that novel TatB–TatB and TatB–TatC contacts, if revealed by treatment with DCCD, might allow new insights into the composition and functioning of TatBC complexes.

      Results

      DCCD-dependent cross-linking reveals a novel TatBC contact site on the membrane surface

      When Escherichia coli membrane vesicles were treated with DCCD and the membrane proteins were subsequently analyzed by SDS-PAGE and immunoblotting, anti-TatB and anti-TatC antibodies both recognized an about 50-kDa protein (Fig. 1A, black dot) in addition to the ∼28-kDa TatB and the ∼23-kDa TatC antigens. (Note that migration of the hydrophobic Tat proteins on SDS gels deviates from their actual molecular masses.) This 50-kDa TatBC-containing protein complex was strictly DCCD-dependent (compare lanes 1 and 2, and 3 and 4, respectively), suggesting that DCCD might be able to cross-link TatB and TatC to form a 1:1 complex. DCCD treatment of E. coli membrane vesicles yielded another prominent species of about 55 kDa (Fig. 1A, lane 2, green star), which, different from the 50-kDa product, was recognized only by anti-TatB antibodies. The 55-kDa product is therefore likely to represent a homodimer of TatB formed by DCCD.
      Figure thumbnail gr1
      Figure 1DCCD couples TatB to TatC. A, inverted inner membrane vesicles of E. coli containing overexpressed levels of TatA, TatB, and TatC (TatABC) were mock-treated with DMSO or treated with 0.5 mm DCCD as indicated. Membrane proteins were resolved by SDS-PAGE and probed by immunoblotting using anti-TatB and anti-TatC antibodies. Hetero- and homodimers of TatB and TatC are labeled. The 40 kDa TatC-cross-reactive band of lane 4 likely represents a homodimer of TatC, whereas the nonlabeled DCCD-dependent cross-links of lane 2 cannot be assigned with certainty. B, SDS-PAGE of 50 μg each of TatBC complex purified via a C-terminal His tag on TatC following either incubation with 0.5 mm DCCD or mock treatment with DMSO (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ).
      Next, we purified the TatBC complex from membrane vesicles via a His tag on TatC, which allows the copurification of TatB when membrane vesicles are solubilized with N-lauroyl sarcosine (Fig. 1B, left lane). If the vesicles were treated with DCCD prior to solubilization, SDS-PAGE revealed three distinguishable 40–55-kDa species co-purifying with the TatB and TatC monomers (Fig. 1B, Dimers 1–3). By size and immunoreactivity, these bands correspond to TatB dimers (1), 1:1 TatBC complexes (2), and TatC dimers (3). To determine the nature of these DCCD-dependent dimers, we subjected all bands marked by arrows in Fig. 1B to proteolytic digestion using trypsin alone or in combination with chymotrypsin and analyzed the resulting peptide mixtures by LC-tandem MS (LC-MS/MS).
      According to the NMR structure of E. coli TatB (Fig. 2A), its first 100 amino acids adopt a mostly α-helical conformation in membranes and membrane-mimetic environments, such that an APH is rigidly packed against the preceding short TM and is followed by the more flexibly arranged helices α3 and α4 (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ). Downstream of this helical region of TatB, the remaining about 70 amino acids are predicted to be largely unstructured. As illustrated in Fig. 2B, the peptides that we identified by LC-MS/MS from the monomeric TatB band shown in Fig. 1B covered almost the entire amino acid sequence of TatB, except for the N-terminal 13 residues including the major part of the TM of TatB. Consistent with the helical part of TatB being exposed to a hydrophobic environment, the lipophilic compound DCCD modified predominantly carboxyl side chains located in that stretch of E. coli TatB. Thus, numerous peptides carrying the dicyclohexylurea (DCU) moiety at either an aspartyl or glutamyl side chain were obtained from DCCD-treated TatB, starting with Glu49 (Fig. 2B). These peptides are highlighted by the blue vertical bars at the positions of the DCU-modified residues, with the lengths of the bars reflecting the relative abundancies of detection. TatBE49 is a conserved residue located in the linker region between APH and helix α3 (Fig. 2A). Its modification by DCCD occurred with one of the highest frequencies (Fig. 2B).
      Figure thumbnail gr2
      Figure 2DCCD-sensitive carboxyl side chains of E. coli TatB. A, NMR structure of the first 101 residues of E. coli TatB (Protein Data Bank code 2MI2) (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ). Highlighted are the TM and APH helices and helices 3 and 4 (α3 and α4) as well as residues addressed in this study. The membrane boundaries were sketched according to the NMR data (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ). B, sequence coverage and sites of modification by DCCD of E. coli TatB determined by in-gel digestion of monomeric TatB using trypsin and chymotrypsin followed by LC-MS/MS analysis. For each amino acid of TatB from 14 to 171, MS intensities of peptides containing this residue were summed up and plotted against its sequence residue number. Relative quantification of peptides was performed based on detected ion intensities. Cumulative intensities of peptides identified with modification of aspartic or glutamic acid by DCU are plotted at positions of the modified sites (diamonds on vertical lines) according to the same logarithmic scale as the total sum of intensities including modified and unmodified peptides. Potential cleavage sites of trypsin and chymotrypsin (black) as well as actual cleavage sites observed in DCCD-treated (blue) and nontreated (green) TatB are indicated in the lower graph. C, vesicles were prepared from Δtat E. coli strains overexpressing TatABC, TatABD3A,E49AC, TatABE8A,E49AC, and TatAC. They were treated with either DCCD or DMSO before incubating with the fluorescent DCCD analog NCD-4. Proteins were separated by SDS-PAGE and analyzed under UV light. The gel was run without molecular mass standards because they could not be visualized using this technique of detection. The identity of the TatB band is indicated by its appearance being affected when using distinct TatB variants. Labeling of TatC by NCD-4 has been addressed previously (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ). D, Western blot analysis using anti-TatB and anti-TatC antibodies to control for expression levels.
      Residues Asp3 and Glu8 of E. coli TatB are the only negatively charged amino acids upstream of Glu49. Because residues 1–13 were not detected in our LC-MS/MS analyses, we addressed the susceptibility of TatBD3 and TatBE8 to a modification by DCCD using labeling of membrane vesicles with the fluorescent analog of DCCD, N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide (NCD-4). As shown in Fig. 2C, NCD-4 stained WT TatB if not antagonized by a 10-fold molar excess of unlabeled DCCD (lanes 1 and 2). Substitution of Glu49 by alanine (TatBE49A) still allowed labeling with NCD-4 (lane 3), which is consistent with the numerous DCCD target sites of TatB depicted in Fig. 2B. However, if Glu8 had also been exchanged against alanine, staining of this TatBE8A/E49A variant with NCD-4 in general was found to be decreased compared with WT TatB (lane 7). This was not due to a lack of expression of the tatBE8A,E49A double mutant (Fig. 2D, lane 4), although the double mutation led to an aberrant running behavior on SDS gels. These results might therefore hint toward the conserved Glu8 of TatB also being a target of DCCD, although we could not rigorously confirm this by other experimentation.
      Next we searched for those DCCD-sensitive carboxyl side chains of TatB that DCCD would cross-link through amide bonds to free amino groups of nearby TatC residues, thereby generating the TatBC heterodimers shown in Fig. 1A. To this end, we screened the entire LC-MS/MS data sets derived from all bands highlighted in Fig. 1B (arrows) for spectra matching branched peptides composed of TatB and TatC peptide sequences. Because the transmembrane helix of TatB, which has been established as a hot spot for intramembrane contacts with TatC (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jager F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • et al.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ,
      • Kneuper H.
      • Maldonado B.
      • Jäger F.
      • Krehenbrink M.
      • Buchanan G.
      • Keller R.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
      ), was not completely represented by our peptide identifications, any DCCD-mediated TatBC cross-link detected by our MS analyses was likely to reveal a new contact site between both proteins.
      All branched peptides identified after digestion with trypsin and containing TatC-derived peptide sequences are summarized in Fig. 3A (extracted from Table S1, positions 403–456). As expected, virtually all of them were derived from the dimer bands highlighted in Fig. 1B. On the TatB side, most DCCD-mediated amide bonds clustered around the glutamyl residues 49, 53, 58. These glutamates specifically formed ω peptide bonds with the α-amino group of serine 2, constituting the N terminus in our TatC preparations, and isopeptide bonds with the ϵ-amino group of lysine 239 located in the C-terminal domain of TatC (Fig. 3B, all lysyl residues of E. coli TatC highlighted by red dots). The most frequent cross-links were those between the N terminus of TatC and the glutamyl residues 49 and 53, which are both located in the linker region connecting the APH with helix α3 of TatB. Thus, in our isolated TatBC complex, this negatively charged, conserved (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ), and according to the NMR structure exposed (Fig. 2A) area of TatB must be positioned in immediate vicinity to the very N terminus of TatC, indicating hitherto unrecognized contacts between TatB and TatC on the membrane surface.
      Figure thumbnail gr3
      Figure 3DCCD-mediated cross-links between TatB and TatC. A, listed are the sites of E. coli TatB and TatC that were found cross-linked via DCCD. The data were extracted from . The terms “monomeric TatC” and “TatBC dimers” refer to B. Frequency of identification reflects the number of cross-linked peptides identified by LC-MS/MS analyses (). B, illustration of the TatBC cross-links given in A. The four helices of TatB (TM, APH, α3, and α4) and the six TMs of TatC are indicated. Red dots, positions of lysyl residues of TatC.
      Cross-linking of the Glu49, Glu53, and Glu77 residues of TatB to both the N terminus of TatC and Lys239, located in the C-terminal domain of TatC, could reflect a close proximity of both ends of TatC. Experimental evidence for such a conformation is provided by the intramolecular TatC cross-link that DCCD formed between Glu244 and Ser2 (Fig. 3A) and by our previous analysis (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ). Of note, the DCCD-mediated cross-links between the N-terminal TatCS2 and the C-terminal TatCE244 were only obtained from monomeric TatC (Fig. 3A), indicating that within a TatBC complex, both surface-exposed ends of each TatC monomer in fact touch each other and do not swing out to connect with neighboring TatC protomers. This is obviously different from TatC oligomers lacking TatB, in which situation the N-terminal TatC domains were found to be highly mobile (
      • Ramasamy S.
      • Abrol R.
      • Suloway C.J.
      • Clemons Jr., W.M.
      The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.
      ,
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jager F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • et al.
      Structure of the TatC core of the twin-arginine protein transport system.
      ).
      The results presented thus far indicate that at least a fraction of those TatB molecules that copurify with TatC must be linked to TatC in a manner that positions the Glu49/Glu53–containing linker region of TatB so closely to the N terminus of TatC that DCCD can form amide bonds between either glutamyl residue of TatB and the free α-amino group of a TatC monomer. To verify this novel TatBC contact site by independent experimental evidence, we replaced TatBE49 and TatCS2 each by cysteines in an otherwise Cys-free TatABC background. When E. coli membrane vesicles carrying these variants of TatB and TatC were treated with either the sulfhydryl-specific cross-linker bismaleimidoethane (BMOE) or the disulfide-forming oxidant sodium tetrathionate (NaTT), TatBC heterodimers of ∼50 kDa were in fact produced, as shown by immunoblotting using antibodies against TatB and TatC (Fig. 4A, black dot). The fact that, compared with BMOE (spacer arm length 8 Å), the zero-space disulfide cross-linker NaTT was not considerably less efficient in generating TatBC heterodimers (compare lanes 3 and 4) would be a further indication of the close molecular proximity of TatBE49 and TatCS2 in the TatBC complex. Fig. 4B (left) illustrates how this novel TatBC contact site would fit into a model of how TatB molecules intercalate between two neighboring TatC monomers as a basis for the generation of circular TatBC structures (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ).
      Figure thumbnail gr4
      Figure 4TatB Glu49 contacts both the N terminus of TatC and Lys93 of a neighboring TatB molecule. A, membrane vesicles of E. coli containing TatA and the indicated cysteine variants of TatB and TatC in a Cys-less TatC background (TatABC ΔCys) were either mock-treated, alkylated using IAA, or incubated with the Cys cross-linkers BMOE and NaTT. Membrane proteins were resolved by SDS-PAGE and probed by immunoblotting using anti-TatB and anti-TatC antibodies. TatB-immunoreactive bands of the size of 2 × TatB and TatB × TatC, which were obtained even in conditions that block the formation of disulfides (lane 1, DTT; lane 2, IAA), probably represent background material. The TatC-immunoreactive band labeled with a blue star corresponds in size to a dimer of TatC. Sizes of marker proteins (M) are given. B, models of how TatB intercalating between two TatC monomers promotes circular TatBC arrays. The suggested position of TatB is supported by the established contact between TatBE49 and the N terminus of TatC (left; the RR-recognition site of TatC consisting of its N-terminal domain and TM 2/TM 3 loop lies behind the Glu49-proximal end of the TatB APH). This position of TatB is also consistent with two neighboring TatB monomers interacting with each other via Glu49 and Lys93 (right). TatC is represented by its six numbered TMs, and TatB is represented only by its TM and APH (left) or by its four helices (right). C, as in A using vesicles with the indicated Cys variants of TatB and TatC. All lanes shown on either of the two blots are derived from a single SDS gel each. Two of the blotted lanes showing results of an additional mutant were excised between lanes 6 and 7. To visualize stained molecular mass standards, which had been run to the left of the lane labeled 1, the blot was first photographed before exposing it to the antibodies. This lane was aligned with the decorated blots.

      Multiple intra- and intermolecular TatB contacts detected by DCCD-dependent cross-linking

      Cysteine cross-linking of TatBE49C/TatCS2C membrane vesicles, in addition to yielding TatBC heterodimers, also resulted in the formation of TatB homodimers (Fig. 4A, green star). To pursue this further, we employed vesicles that contained the TatBE49C single mutation. The addition of BMOE to these vesicles caused the same TatB dimer formation as observed for TatBE49C/TatCS2C vesicles (Fig. 4C, left, lanes 4 and 6, green star), whereas TatBC heterodimers were not obtained from the TatBE49 vesicles due to the missing TatCS2C alteration (Fig. 4C, both panels, lanes 4 and 6, black dot). Homodimerization of TatB in the TatBE49C/TatCS2C and the TatBE49C vesicles therefore occurs through disulfide bonds between the Glu49 residues of two closely spaced TatB monomers, similar to what had previously been reported for numerous TatB residues upstream of Glu49 when replaced by cysteines (
      • Lee P.A.
      • Orriss G.L.
      • Buchanan G.
      • Greene N.P.
      • Bond P.J.
      • Punginelli C.
      • Jack R.L.
      • Sansom M.S.
      • Berks B.C.
      • Palmer T.
      Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.
      ).
      Disulfide bonding between identical residues of two TatB monomers invokes their parallel, side-by-side orientation. We reasoned that if this indeed were the case, DCCD might cause the formation of isopeptide bonds between closely spaced carboxyl and amino groups of two neighboring TatB polypeptide chains. We therefore screened the LC MS/MS-derived peptide spectra for matches to branched peptides containing only TatB sequences. As summarized in Fig. 5A, an unexpectedly large number of branched peptides was obtained after digesting the monomeric and dimeric TatB bands shown in Fig. 1B with trypsin (for detailed information, see Table S1, positions 1–402). However, a merely superficial glance at the residues of TatB that became cross-linked by DCCD reveals that except for the Glu49-Lys55 and Glu90-Lys93 pairs (Fig. 5B, red lines) all other DCCD-catalyzed isopeptide bonds had formed between residues that are too distant to explain them by a parallel alignment of two TatB monomers.
      Figure thumbnail gr5
      Figure 5Intra- and intermolecular TatB cross-links mediated by DCCD. A, summary of the DCCD-generated branched TatB peptides. The data were extracted from . The terms “monomeric Tat” and “dimeric TatB” refer to B. B, illustration of the major TatB–TatB contacts of A along the TatB sequence. Thick lines represent the DCCD-mediated isopeptide bonds most frequently observed. Red lines mark those bonds that could result from parallel, side-by-side arranged TatB monomers; all others require either an offset alignment of two TatB monomers or reflect a highly mobile C terminus of TatB, as suggested in C.
      In detail, over 80% of all DCCD-mediated TatB–TatB cross-links originated from the two glutamates Glu49 and Glu53 (Fig. 5A). In the vast majority of cases, these glutamates were found cross-linked to the two lysines Lys93 and Lys103 located at the distal end of helix α4 (Fig. 5B, thick blue lines). A smaller fraction of Glu49/Glu53-binding partners was represented by the more proximal lysines Lys65 and Lys68 within helix α3 of TatB (Fig. 5B, blue lines). Clearly, all of these contacts would require either a permanent or temporarily folded conformation of the TatB polypeptide chain or, alternatively, a crossover arrangement of two TatB monomers. For example, if the linker region connecting helices α3 and α4 allowed both helices to pack against each other, the residues Lys93/Lys103 would come into close proximity to the Glu49/Glu53 area of TatB (Fig. 5C). In fact, the data collection of Fig. 5A reveals that one-third of the branched peptides, in which Glu49 was found cross-linked to Lys93/Lys103, were recovered from the monomeric TatB band of Fig. 1B. Even more frequently, the branched peptides originating from cross-links between Glu53 and Lys93/Lys103 were recovered from the TatB monomer than from the dimer (Fig. 5A). These intramolecular contacts demonstrate that helix α4 of TatB can form hairpin structures with preceding parts of the molecule, as depicted in Fig. 5C, which would be consistent with the high mobility of helix 4 observed in previous NMR studies (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ).
      Despite this considerable number of intramolecular TatB cross-links obtained by treatment with DCCD, the majority of the cross-links between Glu49 and Lys93/Lys103 were, however, recovered from the dimer bands of Fig. 1B, as the data presented in Fig. 5A reveal. In other words, Glu49 predominantly formed intermolecular contacts with the Lys93 and Lys103 residues of a neighboring TatB monomer. These cross-links actually represent the largest fraction of all DCCD-mediated TatB–TatB contacts detected (Fig. 5A). They suggest that a considerable fraction of the TatB molecules copurifying with TatC adopt a nonparallel, branched orientation such that TatB monomers intersect at their Glu49 and Lys93/Lys103 residues.
      If, for example, two TatB monomers became cross-linked via the side chains of Glu49 and Lys93, both coupled monomers would still retain a free Lys93 and a free Glu49 residue, respectively, to form cross-links with two more adjacent TatB monomers. Continuation of this process would lead to TatB oligomers covalently linked through their Glu49/Lys93 sites. To address such an oligomerization of TatB experimentally, we constructed a TatB variant carrying both the E49C and K93C alterations. If membrane vesicles obtained from this tatB double mutant were treated with the sulfhydryl-specific cross-linker BMOE, a considerable fraction of TatB-immunoreactive molecules displayed sizes much larger than that of a TatB dimer (Fig. 4C, left, lane 7). We therefore conclude that the major fraction of TatB molecules copurifying with TatC are arranged in an oligomeric manner such that their Glu49 residues are positioned in the immediate vicinity of the distal end of helix α4 of an adjacent TatB monomer (Fig. 4B, right). These novel intermolecular TatB contacts would thus support the idea of circular TatBC receptor structures, which has been put forward on the basis of distinct contact sites between TatB and TatC (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ), TatC and TatC (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ), TatB and TatB (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ), and TatB and substrate (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ,
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ).

      TatB improves the formation of a functional substrate-binding site on TatC

      The data presented thus far indicate that when TatB is bound to TatC, the Glu49/Glu53 linker region of TatB is predominantly found in contact both with downstream regions of TatB as well as with the substrate-binding site of TatC. The latter is composed of the N-terminal domain and the juxtaposed TM 2/TM 3 loop of TatC (
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ,
      • Strauch E.M.
      • Georgiou G.
      Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides.
      ). Therefore, the question arose of whether the negatively charged Glu49/Glu53 linker region of TatB was directly involved in substrate recognition and binding by the TatBC complex.
      As demonstrated several times before (
      • Eimer E.
      • Kao W.C.
      • Fröbel J.
      • Blümmel A.S.
      • Hunte C.
      • Müller M.
      Unanticipated functional diversity among the TatA-type components of the Tat protein translocase.
      ,
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alami M.
      • Lüke I.
      • Deitermann S.
      • Eisner G.
      • Koch H.G.
      • Brunner J.
      • Müller M.
      Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.
      ), binding of a Tat substrate to the TatBC complex of membrane vesicles can be visualized by the use of Tat precursor proteins that carry the photoactivatable cross-linker p-benzoylphenylalanine (Bpa) in their signal sequences. Thus, Bpa engineered at position Phe14 in the RR-consensus motif of the model Tat precursor protein TorA-mCherry (TmC) cross-links to TatC of TatABC-containing vesicles if activated by UV light (Fig. 6A, lanes 1 and 2, blue star). In contrast, when Bpa is positioned at position Leu27 in the hydrophobic core of the signal sequence, it yields cross-links to TatB and TatA (lanes 7 and 8, green and pink stars). In vesicles that carried an alanine exchange of TatBE49, both Bpa-containing variants of TorA-mCherry showed an unimpaired association with TatC, TatB, and TatA (lanes 2, 4, 8, and 10; the differing sizes of the TatC-TmC adducts of lanes 2 and 4 are explained by a His tag on the TatC of the control vesicles). Only when Glu49 and Glu53 of TatB were simultaneously replaced by alanine did cross-linking to TatA, TatB, and TatC seem to be diminished (lanes 6 and 12). The failure to cross-link to TatB can be explained by the drastically reduced level of TatB that we repeatedly observed for vesicles carrying the TatBE49A/E53A variant (Fig. 6B, lane 3). Obviously, the simultaneous loss of both glutamyl residues Glu49 and Glu53 causes TatB to become unstable, most likely because of an impaired association with TatC and neighboring TatBs. In contrast to TatB, the level of TatC was not substantially altered in the TatBE49A/E53A vesicles (Fig. 6B, lane 3), and yet, precursor binding to TatC was strongly diminished (Fig. 6A, lanes 4 and 6). Whereas these cross-linking data do not support a direct involvement of the Glu49/Glu53 linker region of TatB in substrate binding, they nevertheless suggest that proficient precursor binding to TatC depends on the presence of TatB.
      Figure thumbnail gr6
      Figure 6Requirement of TatB for substrate recognition by TatC. A, autoradiography. Two variants of the Tat precursor (p) protein TmC with Bpa incorporated at positions Phe14 and Leu27 were synthesized and radioactively labeled in vitro. Control membrane vesicles (TatABC ΔCys) and vesicles harboring the indicated alanine exchanges in TatB were added. Cross-linking was initiated by irradiation with UV light. Marked are adducts of pTorA-mCherry to TatA, TatB, and TatC. B, contents of TatB and TatC of the vesicles as determined by immunoblotting with anti-TatB and anti-TatC antibodies. C, as in A, comparing cross-linking of pTorA-mCherry to TatC, TatB, and TatA in the presence (TatABC) and absence (TatAC) of TatB. Control samples with no vesicles added are shown in lanes 1, 2, 7, and 8. D, TatC content of the vesicles used in C as determined by immunoblotting against TatC.
      To directly demonstrate this, we compared binding of TorA-mCherry to membrane vesicles in the presence and absence of TatB (Fig. 6C). As expected, in vesicles harboring only TatA and TatC, cross-linking between the L27Bpa variant of TorA-mCherry and TatB was missing (compare lanes 10 and 12). As reported previously (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ), in the absence of TatB, Leu27 of TmC was found in proximity to TatC (lane 12). TatAC vesicles, however, showed an impaired cross-linking also of the F14Bpa variant of TmC to TatC (compare lanes 4 and 6), although their TatC content was even higher than that of TatABC vesicles (Fig. 5D). A possible explanation for this finding is that an efficient formation of the substrate recognition site from the two known epitopes of TatC, its N-terminal domain and the TM 2/TM 3 loop, depends on the presence of TatB. Through juxtaposition to the Glu49/53 linker region of TatB, the per se flexible N-terminal domain of TatC (
      • Ramasamy S.
      • Abrol R.
      • Suloway C.J.
      • Clemons Jr., W.M.
      The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.
      ,
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jager F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • et al.
      Structure of the TatC core of the twin-arginine protein transport system.
      ) might become more stably positioned next to the TM 2/TM 3 loop.

      Lateral access of TatA to TatBC-bound substrate

      It is experimentally well-established (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jager F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • et al.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ,
      • Habersetzer J.
      • Moore K.
      • Cherry J.
      • Buchanan G.
      • Stansfeld P.J.
      • Palmer T.
      Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.
      ,
      • Kneuper H.
      • Maldonado B.
      • Jäger F.
      • Krehenbrink M.
      • Buchanan G.
      • Keller R.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
      ) that TatB and TatC also contact each other within the lipid bilayer through the TM of TatB and the TM 5 of TatC. The latter is part of the substrate-binding cavity in the interior of the TatBC complex (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ). Recent data suggest that upon substrate binding, the TM of TatB shifts its position from TM 5 of TatC to TM 6 and that this shift goes along with the reciprocal recruitment of TatA from the outside position on TM 6 to TM 5 of TatC (
      • Habersetzer J.
      • Moore K.
      • Cherry J.
      • Buchanan G.
      • Stansfeld P.J.
      • Palmer T.
      Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.
      ). This position switching between TatB and TatA would require a movement of the TatA and TatB TMs in between two neighboring TatC monomers. We tried to address this experimentally by cross-linking the two residues, Phe69 and Ala133, of TatC that are predicted to flank the path of exchange of the TatA and TatB TMs (Fig. 7A). When these residues were each replaced by cysteines, the addition of the cross-linker BMOE caused a considerable formation of dimers, tetramers, and hexamers of TatC (Fig. 7B), providing evidence for the molecular proximity of Phe69 and Ala133 in the oligomeric TatBC complex. The simultaneous F69C and A133C substitutions of TatC allowed for a reduced, yet still substantial, translocation activity into membrane vesicles, as assayed by the amounts of proteinase K (PK)-resistant precursor (p) and mature form (m) of the Tat precursor protein SufI (Fig. 7C, compare lanes 1 and 2 with lanes 5 and 6). The addition of BMOE, however, strongly suppressed translocation (lanes 7 and 8). This result indicates that a covalent linkage of two neighboring TatC protomers within the oligomeric TatBC complex is incompatible with a functional Tat translocase.
      Figure thumbnail gr7
      Figure 7Lateral fixation of TatC monomers interferes with TatA contacts and transport of RR precursors. A, detail of a TatB molecule intercalated between two TatC monomers that approximate each other via the Ala133 and Phe69 residues. B, vesicles containing either WT TatA, TatB, and a cysteine-less variant of TatC (ΔCys) or a TatCF69C/A133C double cysteine variant of an otherwise cysteine-less TatC were treated with BMOE where indicated. C, autoradiography. SufI was synthesized and radioactively labeled in vitro. Vesicles were pretreated with BMOE as indicated and added to the in vitro reaction. Translocation is demonstrated via processing of the precursor (p) to the mature form (m) and proteinase K (PK) resistance. Transport efficiency was quantified from three independent experiments. D, autoradiography. TmC harboring Bpa at position Leu27 was synthesized and radioactively labeled in vitro. Vesicles were pretreated with BMOE as indicated and added to the in vitro reaction. Samples were subsequently irradiated with UV light. Green and pink stars mark adducts of TmC to TatB and TatA, respectively. The amounts of TmC interacting with TatB and TatA were each quantified relative to the amounts of TmC synthesized. The adducts obtained in lane 2 were then set to 100%. Values are derived from four independent experiments. E, autoradiography. TorA-mCherry was synthesized and radioactively labeled in vitro. To deplete ATP from the samples, hexokinase and glucose (Hex/Glu) were added after synthesis. NADH was added to restore the PMF in the absence of ATP. Subsequently, TatABC vesicles containing the TatCE170A variant were added, and transport was tested by proteinase K resistance of the precursor and mature form. After proteinase K treatment, the precursor is of slightly reduced size because of proteinase K–mediated removal of a few N-terminal amino acids. F, TmC harboring Bpa at position Leu27 was synthesized and radioactively labeled in vitro. After synthesis, ATP was depleted, and vesicles containing the TatCE170A mutation were added together with NADH. After incubation with vesicles, samples were irradiated with UV light. DCCD was added as indicated prior to the vesicles. Green and pink stars, adducts of TmC to TatB and TatA, respectively.
      We then investigated the association of the Tat precursor protein TorA-mCherry with membrane vesicles carrying the TatCF69C/A133C double variant. This was done via Bpa-dependent cross-linking using TmC with Bpa incorporated at position Leu27 of the TorA signal peptide (Fig. 7D, TmC L27-Bpa). Compared with control vesicles harboring cysteine-less TatC (lane 2), the percentage of cross-linked TmC L27-Bpa precursor molecules to TatB and TatA of the TatCF69C/A133C vesicles (lane 5, green and pink stars) was reduced, when corrected for the differing input levels of TmC L27-Bpa. This would be consistent with the reduced transport activity of those mutant vesicles (Fig. 7C, lanes 5 and 6). However, whereas the addition of BMOE to TatCF69C/A133C vesicles did not significantly change cross-linking to TatB (green star, compare lanes 5 and 6), it caused a substantial decrease in precursor contacts with TatA (pink star). This suggests a lower availability of TatA after covalently linking adjacent TatC protomers through F69C and A133C. The residual precursor contacts with TatA agree with an incomplete conversion of monomeric TatC into oligomers by BMOE (Fig. 7B) (i.e. incomplete concatenation of TatC) and explain the residual translocation activity under these experimental conditions (Fig. 7C, lanes 7 and 8).
      Because DCCD leads to numerous cross-links among TatB and TatC molecules, it was not unlikely to thereby also influence the access of TatA to the TatBC-embedded signal peptide. When testing this, we, however, had to exclude alternative inhibitory effects of DCCD. As previously shown (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ), DCCD modifies Glu170 of TatC and thereby interferes with the hairpin insertion of a Tat signal peptide into the TatBC-binding cavity. The defective insertion is, however, largely prevented by using membrane vesicles that carry the TatCE170A variant. Furthermore, DCCD inactivates the F1FO-ATPase of our membrane vesicles that is required for generating a PMF, without which TatA does not contact TatBC-inserted precursor proteins. Therefore, we energized our membrane vesicles not via ATP hydrolysis by their F1FO-ATPase but by generating the PMF through oxidation of NADH (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). This is illustrated in Fig. 7E by analyzing the transport of TorA-mCherry into TatABCE170A-containing vesicles. The amounts of proteinase K–resistant precursor (p) and mature form (m) of TorA-mCherry (lane 2) were drastically reduced when the ATP pool of the in vitro reaction was depleted by the addition of hexokinase in combination with glucose (lane 4) but could largely be restored by the addition of NADH (lane 6). In analogy, the PMF-sensitive contact between TatA and the signal peptide of TorA-mCherry L27-Bpa largely disappeared after ATP depletion (Fig. 7F, lanes 2 and 3, pink star) and could be re-installed by NADH (lane 4). If, however, under these PMF-generating conditions, DCCD was added, the precursor–TatA contact was not established (lane 5). Fig. S1 verifies the NADH-dependent generation of an H+-gradient in membrane vesicles treated with DCCD. Thus, DCCD prevents TatA from contacting the Tat substrate much like disulfide bridges between two neighboring TatC molecules do (Fig. 7D). Collectively, these results strongly suggest that to contact the Tat substrate, TatA enters the TatBC-binding cavity through lateral gates between TatBC protomers.

      Discussion

      One major secret yet to be disclosed about the functioning of Tat translocases is the interplay between the apparently disparate TatC- and TatA-type proteins. Knowledge of their molecular interactions might be key to understanding how they cooperate in allowing folded proteins to cross cellular membranes. Although individual structures of TatC and TatA/TatB have been published, structural information on complexes formed from the Tat subunits is missing. This is particularly true for the association of TatB with TatC, both of which have long been known to concertedly form a substrate receptor complex. Because TatA, which is required for the actual translocation of the substrate protein, is a structural homolog of TatB, it will bind to TatC in a similar way as TatB, although the consequence of this interaction would be translocation rather than substrate recognition and binding.
      One approach of identifying contact sites between two proteins is chemical cross-linking. Here we have explored the potential of DCCD to form isopeptide bonds between adjacent proteins. In this way, we discovered novel contact sites between TatB and TatC that do not involve the transmembrane parts of both proteins but rather domains assumed to be attached to the membrane surface. The area around the conserved glutamyl residue Glu49 of E. coli TatB was thus found in immediate proximity to the N- and C-terminal ends of TatC. TatBE49 being located in the linker region between the APH and helix 3 is likely to be positioned on the surface of the lipid bilayer. Its vicinity to either end of TatC is consistent with the previously described molecular proximity of the N-terminal and C-terminal domains of TatC to each other and to the TM 2/TM 3 loop on the cis-surface of the membrane (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ).
      Such a conformation of both terminal domains of TatC excludes the possibility that they could function as permanent connecting points between neighboring TatC monomers in the oligomeric TatBC complexes. Rather, these and previous studies revealed the trans-sided TM 2 residues Asp63 and Phe69 of E. coli TatC associating with the trans-sided TM 4 residue Ala133. These intermolecular TatC contacts are likely to reflect a side-by-side (i.e. head-to-tail) assembly of adjacent TatC monomers as the basis for circular TatC scaffolds of the TatBC receptor complex. Other sites in TatC were found to cause a face-to-face dimerization of TatC (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ,
      • Kneuper H.
      • Maldonado B.
      • Jäger F.
      • Krehenbrink M.
      • Buchanan G.
      • Keller R.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
      ,
      • Cléon F.
      • Habersetzer J.
      • Alcock F.
      • Kneuper H.
      • Stansfeld P.J.
      • Basit H.
      • Wallace M.I.
      • Berks B.C.
      • Palmer T.
      The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.
      ), but it is not clear to what extent these dimers reflect functional units.
      Besides the newly discovered contacts between TatB and TatC on the membrane surface, site-specific cross-linking and sequence co-evolution analysis had shown that both proteins associate with each other within the lipid bilayer through the TM of TatB and residues clustering on TM 5 and 6 and the intervening loop of TatC. Those studies also revealed contacts between TatB and TM 1 and 2 of TatC, which constitute the other flank of TatC opposite to TM 5/6. Thus, the TM of TatB is obviously intercalated between two adjacent TatC monomers, and the continuous packing of one TatB between two TatC monomers would favor the formation of circular TatBC complexes. The concept of circular TatBC complexes again is strongly supported by our novel finding of intermolecular TatB contacts involving the Glu49 and Lys93 residues, thus reinforcing the connection between neighboring protomers by distinct intramembrane TatBC interactions through additional intermolecular TatB contacts on the cytoplasmic membrane surface.
      The highly conserved Glu8 residue of TatB is located at the beginning of the TM of TatB (Fig. 2A). Following covariance analysis and MD simulations, it was recently proposed that residue Glu8 of TatB forms hydrogen bonds with Thr208 (TM 5/6 loop) and Gln215 (TM 6) of TatC (
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ). On the other side, if TatB in fact also contacts TM 2 of TatC, as suggested by Bpa-dependent cross-linking (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ), Glu8 of TatB might even come into close proximity to the TM 2 residue Lys73 of TatC. In principle, the proximity between TatBE8 and TatCK73 could easily be demonstrated by the formation of a DCCD-mediated isopeptide bond, but its verification failed here because of the sequence section of TatB around Glu8 not being covered by our MS data. If TatBE8 and TatCK73 would in fact become juxtaposed at some time during the assembly of the TatBC complex, both residues could form an intramembrane ion bond. Its potential dependence on the local H+ concentration (i.e. the PMF) might even render it a controllable TatBC juncture.
      At least one-third of the DCCD-mediated TatB cross-links involving Glu49/Glu53 and Lys93/Lys103 was intramolecular in nature. This indicates that a considerable fraction of the TatC-associated TatB molecules had adopted a conformation in which helix α4 formed hairpin structures with preceding parts of the TatB molecule (Fig. 5C). If such a compaction of TatB occurred in a TatBC structure, as sketched in Fig. 4B, the Lys93 area of the folded TatB monomer could no longer contact the Glu49 region of a neighboring TatB molecule. In theory, an intermolecular contact might then still be possible between Glu49 of one TatB and Lys30 located at the N-proximal end of a neighboring TatB APH (cf. Fig. 4B). In fact, DCCD-caused Glu49–Lys30 isopeptide bonding was obtained (Fig. 5A), yet with dramatically lower frequency than that between Glu49 and Lys93. An alternative explanation for the DCCD-dependent intramolecular TatB cross-links therefore would be that they reflect the high flexibility of the C terminus of TatB revealed by NMR analysis. In theory, this flexibility could be linked to conformational changes of TatB during substrate binding.
      Disulfide cross-linking of two TatB monomers as a consequence of replacing Glu49 by cysteines (this study) as well as multiple residues within the TM and APH of TatB (
      • Lee P.A.
      • Orriss G.L.
      • Buchanan G.
      • Greene N.P.
      • Bond P.J.
      • Punginelli C.
      • Jack R.L.
      • Sansom M.S.
      • Berks B.C.
      • Palmer T.
      Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.
      ) invokes the existence of TatB subpopulations with an at least partially linear, registered alignment of two or even more TatB molecules. The DCCD-mediated cross-linking of two TatBs through Glu49/Lys55 and Glu90/Lys93 (cf. Fig. 5) suggests that a nearly parallel oligomerization of TatB seems to occur at some frequency even for TatC-associated TatB molecules. At this point, it is not clear whether such oligomers would populate the inside or the periphery of a circular TatBC structure.
      Early Bpa-dependent cross-linking studies had revealed the proximity between TatB and the N-terminal domain of TatC (
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ), which is part of the RR-signal peptide recognition site of TatC (
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ). We have now identified the Glu49/Glu53 linker region of TatB as a contact site for the N terminus of TatC, which in turn comes close to the TM 2/TM 3 loop of TatC (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ) to constitute the RR-recognition site. Despite this molecular vicinity, we were not able to demonstrate a direct involvement of the Glu49/Glu53 linker region of TatB in the substrate recognition process. We found, however, that the interaction of the RR-signal peptide with its recognition site on TatC was disturbed in the absence of TatB. This could mean that the Glu49/Glu53 linker region of TatB functions as a clamp for the N terminus of TatC to secure its juxtaposition to the other RR-interacting epitope of TatC (i.e. the TM 2/TM 3 loop).
      Habersetzer et al. (
      • Habersetzer J.
      • Moore K.
      • Cherry J.
      • Buchanan G.
      • Stansfeld P.J.
      • Palmer T.
      Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.
      ) recently found that overproduction of substrate in whole E. coli cells caused the exchange of TatA for TatB at TM 5 of TatC and a corresponding positional switch of both proteins at TM 6 of TatC. This implies that TatA moves from a more peripheral position around the TM 6 toward the concave face of TatC, which was actually experimentally verified for the chloroplast Tat translocase (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). Our data lend further support to such a functional movement of TatA to the interior of circularly arranged TatC protomers. Thus, we found that contacts between TatA and substrate being a prerequisite for a TatA-mediated translocation event are impaired by locking the lateral entrance of TatA between neighboring TatC protomers. The same effect was achieved by DCCD, which we found to form numerous cross-links between TatB/TatC and TatB/TatB and thereby most likely prevents TatB from vacating the TatA-binding sites on TatC. At this point, however, we cannot exclude the possibility that DCCD, besides cross-linking TatB and TatC to each other, might also influence the contact between substrate and TatA that was reported previously (
      • Taubert J.
      • Hou B.
      • Risselada H.J.
      • Mehner D.
      • Lünsdorf H.
      • Grubmüller H.
      • Brüser T.
      TatBC-independent TatA/Tat substrate interactions contribute to transport efficiency.
      ).
      Subsequent events following the association of TatA with substrate are still hypothetical. Further experimentation will have to address whether the circular arrangement of TatC persists and TatA oligomers form a translocation platform within an expanding TatC ring or whether TatC monomers undergo a substantial reorientation (
      • Habersetzer J.
      • Moore K.
      • Cherry J.
      • Buchanan G.
      • Stansfeld P.J.
      • Palmer T.
      Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.
      ,
      • Cléon F.
      • Habersetzer J.
      • Alcock F.
      • Kneuper H.
      • Stansfeld P.J.
      • Basit H.
      • Wallace M.I.
      • Berks B.C.
      • Palmer T.
      The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.
      ) or whether a TatA pore laterally buds off from a circular TatBC receptor (
      • Alcock F.
      • Stansfeld P.J.
      • Basit H.
      • Habersetzer J.
      • Baker M.A.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Assembling the Tat protein translocase.
      ).

      Experimental procedures

      Plasmids

      Plasmids used in this study are listed in Table S2. Plasmid p8737 was used to introduce the Ala codon GCG into the tatB gene and plasmid pUNITATCC4 (
      • Lee P.A.
      • Orriss G.L.
      • Buchanan G.
      • Greene N.P.
      • Bond P.J.
      • Punginelli C.
      • Jack R.L.
      • Sansom M.S.
      • Berks B.C.
      • Palmer T.
      Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.
      ) to introduce the Cys codon TGT into the tatB and tatC genes according to the QuikChange site-directed mutagenesis kit protocol (Stratagene). All primers used in this study are listed in Table S3. Amber stop codon mutations in the gene encoding TorA-mCherry of plasmid pPJ3 have been described (
      • Blümmel A.S.
      • Haag L.A.
      • Eimer E.
      • Müller M.
      • Fröbel J.
      Initial assembly steps of a translocase for folded proteins.
      ).

      Membrane vesicles

      Inside-out inner membrane vesicles (INV) were prepared as described (
      • Moser M.
      • Panahandeh S.
      • Holzapfel E.
      • Müller M.
      In vitro analysis of the bacterial twin-arginine-dependent protein export.
      ) from E. coli strains BL21(DE3)* (Novagen) or BL21(DE3)ΔTat (kindly provided by B. Ize and T. Palmer) transformed with plasmid p8737 or pUNITATCC4 and derivatives thereof.

      In vitro reactions

      The RR-precursor proteins TorA-mCherry and pSufI were synthesized by in vitro transcription/translation using plasmids pPJ3 and pEJ. Cell extracts used for the in vitro synthesis were prepared (
      • Moser M.
      • Panahandeh S.
      • Holzapfel E.
      • Müller M.
      In vitro analysis of the bacterial twin-arginine-dependent protein export.
      ) from E. coli strain SL119 (
      • Lesley S.A.
      • Brow M.A.
      • Burgess R.R.
      Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies.
      ) or alternatively from Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of TorA-mCherry (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ). Coupled transcription/translation reactions were performed in 50-μl aliquots as described (
      • Moser M.
      • Panahandeh S.
      • Holzapfel E.
      • Müller M.
      In vitro analysis of the bacterial twin-arginine-dependent protein export.
      ). INV were added 10–15 min after starting the synthesis reaction and incubated for 20 min at 37 °C.
      Assaying protein translocation into INV by proteinase K protection and Bpa-dependent cross-linking by irradiating samples with UV light for 20 min on ice have been described (
      • Fröbel J.
      • Rose P.
      • Müller M.
      Early contacts between substrate proteins and TatA translocase component in twin-arginine translocation.
      ). ATP was depleted by incubating samples with 0.02–5 units/ml hexokinase (Sigma) and 12–20 mm glucose for 10 min at 37 °C. The PMF was restored by adding 4 mm NADH. SDS-PAGE using 10% gels was performed as described (
      • Moser M.
      • Panahandeh S.
      • Holzapfel E.
      • Müller M.
      In vitro analysis of the bacterial twin-arginine-dependent protein export.
      ).

      Cross-linking using DCCD

      To detect contacts between the Tat subunits using DCCD, 2 μl of each INV preparation (∼100 A280 units/ml) were diluted with 47.5 μl of INV buffer (
      • Moser M.
      • Panahandeh S.
      • Holzapfel E.
      • Müller M.
      In vitro analysis of the bacterial twin-arginine-dependent protein export.
      ). After adding either 0.5 mm DCCD or 0.5 μl of DMSO, INV were incubated for 10 min at 37 °C and 300 rpm. Proteins were precipitated with 5% TCA and resuspended in 40 μl of SDS-loading buffer. Aliquots of 20 μl were each analyzed by SDS-PAGE (10% gels) and Western blotting using either anti-TatB or anti-TatC antibodies.

      Disulfide cross-linking

      Two μl of each INV preparation (∼100 A280 units/ml) were diluted with 98 μl of DTT-free INV buffer. Disulfide formation was initiated through the addition of either 0.1 mm BMOE or 1 mm NaTT. Control samples were mock-treated with either 20 mm DTT or 10 mm iodactetamide (IAA). Samples were incubated for 30 min at 25 °C. The BMOE reaction was stopped with 20 mm DTT for 5 min at 37 °C. For detecting contacts between the Tat subunits, the treated INV were subsequently precipitated with 5% TCA and resuspended in 42 μl of SDS-loading buffer in the case of NaTT without DTT. Aliquots of 20 μl were each analyzed by SDS-PAGE (10% gels) and Western blotting using either anti-TatB or anti-TatC antibodies. For use in in vitro assays, the BMOE-treated INV were centrifuged at 90 000 × g for 45 min, and the pellet was resuspended in 5 μl of INV buffer containing 20 mm DTT (14,000 rpm, 30 min).

      Purification of DCCD-modified TatB and TatC

      Purification of TatC and of TatC-associated TatB was performed as described (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ).

      LC-MS/MS

      In-gel digestion of proteins was performed essentially as described (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ). In brief, proteins in bands excised from SDS-polyacrylamide gels were destained and subjected to reduction and alkylation of cysteine residues followed by digestion overnight with either trypsin as the sole protease or in combination with chymotrypsin. Peptide mixtures of three biological replicates were analyzed by LC-MS/MS using an UltiMate 3000 RSLCnano coupled to a Q Exactive Plus (Thermo Fisher Scientific) mass spectrometer followed by peptide identification and quantification using the program MaxQuant and identification of cross-linked peptides using the program pLink as described (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ). DCU-modified peptides were identified as variable modification of aspartate or glutamate (+206.17830 Da) and a modification-specific neutral loss of −125.08406 Da. Cross-linked sites were visualized using the program xiNET (version 2.0) (
      • Combe C.W.
      • Fischer L.
      • Rappsilber J.
      xiNET: cross-link network maps with residue resolution.
      ).

      Identification of DCCD-binding sites

      The binding of DCCD was detected directly by MS analysis (see above) or indirectly using the fluorescent DCCD analogue NCD-4 (Synchem) as described (
      • Blümmel A.S.
      • Drepper F.
      • Knapp B.
      • Eimer E.
      • Warscheid B.
      • Müller M.
      • Fröbel J.
      Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide.
      ).

      Author contributions

      J. F., B. W., and M. M. conceptualization; J. F. supervision; J. F., A.-S. B., and F. D. investigation; J. F. and B. W. writing-review and editing; A.-S. B. and F. D. methodology; F. D. formal analysis; F. D. validation; B. W. and M. M. funding acquisition; M. M. writing-original draft.

      Acknowledgments

      We thank Bettina Knapp for excellent technical assistance.

      Supplementary Material

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