title: Mapping cell envelope and periplasm protein interactions of Escherichia coli respiratory formate dehydrogenases by chemical cross-linking and mass spectrometry.
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/photo-reactive 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.
authors.affiliation: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg , Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Zorn
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ihling
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D002473:Cell Wall; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins / Q000737:chemistry / Q000378:metabolism*; D011422:Propionates / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D053719:Tandem Mass Spectrometry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011422:Propionates; D005560:Formate Dehydrogenases
keywords: chemical cross-linking; formate dehydrogenases; mass spectrometry; photoreactive cross-linker; protein−protein interactions