The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Abstract

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed. Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed. Gram-negative bacteria have a cell envelope composed of two membranes, the inner membrane and the outer membrane (OM), and an enclosed space known as the periplasm. The proteins embedded in the OM mediate a variety of physiological functions including protecting bacteria against external environmental stresses and transporting nutrients to maintain cellular metabolism (1Koebnik R. Locher K.P. Gelder P.V. Structure and function of bacterial outer membrane proteins: Barrels in a nutshell.Mol. Microbiol. 2000; 37: 239-253Crossref PubMed Scopus (861) Google Scholar). Almost all outer membrane proteins (OMPs) are anchored in the OM by a “β barrel,” a closed cylindrical structure composed of antiparallel β strands (2Horne J.E. Brockwell D.J. Radford S.E. Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria.J. Biol. Chem. 2020; 295: 10340-10367Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). β barrels vary in size from 8 to 36 β strands, and some OMPs form homodimers or homooligomers (2Horne J.E. Brockwell D.J. Radford S.E. Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria.J. Biol. Chem. 2020; 295: 10340-10367Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 3Lauber F. Deme J.C. Lea S.M. Berks B.C. Type 9 secretion system structures reveal a new protein transport mechanism.Nature. 2018; 564: 77-82Crossref PubMed Scopus (74) Google Scholar, 4Fairman J.W. Noinaj N. Buchanan S.K. The structural biology of β-barrel membrane proteins: A summary of recent reports.Curr. Opin. Struct. Biol. 2011; 21: 523-531Crossref PubMed Scopus (166) Google Scholar). Besides containing a common β barrel domain, some OMPs also have segments that reside inside the β barrel lumen and/or extracellular or periplasmic domains. After newly synthesized OMPs are transported across the inner membrane through the Sec machinery, they interact with a variety of periplasmic chaperones including SurA, Skp, DegP, and OsmY that maintain them in an insertion-competent conformation and facilitate their integration into the OM (5Chen R. Henning U. A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins.Mol. Microbiol. 1996; 19: 1287-1294Crossref PubMed Scopus (156) Google Scholar, 6Lazar S.W. Kolter R. SurA assists the folding of Escherichia coli outer membrane proteins.J. Bacteriol. 1996; 178: 1770-1773Crossref PubMed Google Scholar, 7Rouvière P.E. Gross C.A. SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins.Genes Dev. 1996; 10: 3170-3182Crossref PubMed Scopus (247) Google Scholar, 8Schäfer U. Beck K. Müller M. Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 9Krojer T. Sawa J. Schäfer E. Saibil H.R. Ehrmann M. Clausen T. Structural basis for the regulated protease and chaperone function of DegP.Nature. 2008; 453: 885-890Crossref PubMed Scopus (282) Google Scholar, 10Yan Z. Hussain S. Wang X. Bernstein H.D. Bardwell J.C.A. Chaperone OsmY facilitates the biogenesis of a major family of autotransporters.Mol. Microbiol. 2019; 112: 1373-1387Crossref PubMed Scopus (10) Google Scholar). These periplasmic chaperones play not only partially redundant roles but also distinct roles in OMP biogenesis. Skp is a jellyfish-like homotrimer that can accommodate unfolded or partially folded OMPs in its central cavity to protect them from misfolding in the aqueous environment (11Walton T.A. Sousa M.C. Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation.Mol. Cell. 2004; 15: 367-374Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 12Korndörfer I.P. Dommel M.K. Skerra A. Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture.Nat. Struct. Mol. Biol. 2004; 11: 1015-1020Crossref PubMed Scopus (111) Google Scholar). Skp either transfers OMPs to other chaperones such as SurA or identifies defective OMPs and directs them to proteases for degradation (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar, 14Combs A.N. Silhavy T.J. The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2114997119Crossref PubMed Scopus (1) Google Scholar). SurA targets OMPs to a heteroligomer known as the barrel assembly machinery (Bam) complex that catalyzes their insertion into the OM. The Bam complex consists of a highly conserved subunit (BamA) that contains a 16-stranded β barrel integrated into the OM and five globular polypeptide in the periplasmic space as as a of that to the T. J. N. S. Silhavy T.J. of a complex required for outer membrane biogenesis in Escherichia Full Text Full Text PDF PubMed Scopus Google Scholar, S. J.C. Silhavy T.J. Structure and function of an of the outer membrane protein assembly PubMed Scopus Google Scholar). of the is conserved and for J.C. J. S. R. Silhavy T.J. the complex and is for outer membrane protein assembly in Escherichia Microbiol. PubMed Scopus Google Scholar, J.C. Silhavy T.J. of outer membrane β-barrel proteins: The 2011; PubMed Scopus Google Scholar). the structure of the Bam complex been A.N. Brockwell D.J. 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A. 2018; PubMed Scopus Google Scholar). in the β barrel is in a closed that is for function and to different models for OMP In the it that OMPs into the as fully unfolded proteins and fold into β that into the of the membrane through a the of β barrel. In an it that the of the barrel the lipid bilayer to the insertion of folded or partially folded OMPs into the OM Sousa M.C. in the β-barrel PubMed Scopus Google Scholar, membrane β-barrel protein folding is by periplasmic lipid and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A.N. Brockwell D.J. Radford S.E. of periplasmic chaperones and membrane protein Mol. Biol. PubMed Scopus Google Scholar). to between an OMP assembly and Bernstein H.D. Bacterial outer membrane proteins with the 2019; 10: PubMed Scopus Google Scholar). that a barrel with an OMP through a between and the C-terminal β strand of the segment that contains the conserved “β is an (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google and a between and N-terminal of the Bernstein H.D. Bacterial outer membrane proteins with the 2019; 10: PubMed Scopus Google Scholar). with the a structure of the assembly to the Bam complex by the of with the β and the of the the other of the barrel T. M. J.E. Bernstein H.D. structures reveal of bacterial outer membrane protein 2022; Full Text Full Text PDF PubMed Scopus Google Scholar). an new in the β binds to and catalyzes the insertion of the of the barrel by a the not fully the structure of the protein before it is integrated into the OM. To help the folded of a β barrel to its insertion into the OM and insight into the of OMP we an but observation that reported over 20 years In an of Escherichia coli OmpA, a and highly OMP that contains an N-terminal β barrel and a C-terminal periplasmic domain, R. 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Full Text PDF PubMed Google Scholar). a OmpA polypeptide that to the β barrel observed in the of but only when and we only a of of the in the periplasm. the results show that OmpA folds into a functional β barrel but only the that and interact to form a native folding in vivo but into β we that if disulfide bonds that the can be the efficiency of assembly To we of residues aligned in β strands in the fully folded OmpA β barrel structure to cysteine In the structure of fully folded E. coli OmpA the and T. J.H. the of structures of membrane proteins by of structure of outer membrane protein Chem. PubMed Scopus Google and the structure of the E. coli OmpA J. R. A. T. M. K. R. A. A. A. A. protein structure with 2021; PubMed Scopus Google all the residues are in the barrel The between the two in is to and is the required for formation X. X. X. 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A. new selectively Gram-negative 2019; PubMed Scopus Google the assembly of In we with a that and or a of cysteine the of a regulated the cell of with the other The folding of OmpA by the and of the folded of OmpA to observed in in folded protein be when In only of the OmpA fragment observed in but we to a molecular protein as a to show that of cell in These results that is required to the insertion of OmpA into the OM and to maintain its in the periplasm. to obtain that the Bam complex an in the assembly of OmpA, we into the β in to its with the conserved residues in the β of and and to The to the the of the OmpA β barrel to the Bam complex and increase its to proteases (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar). the of β the folding of OmpA cysteine in by in the β of and greatly the folding efficiency of OmpA and protein degradation A and a β and assembly A and the of a protein as a we that of cell in and the observation that the of the periplasmic chaperone Skp the degradation of OMPs that contain β and their assembly (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar, 14Combs A.N. Silhavy T.J. The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2114997119Crossref PubMed Scopus (1) Google we to if the of the OmpA would increase in the assembly of the the folded form of the and These further that OmpA is linked by disulfide bonds in the the protein is assembled by the to the as native In we the observation in that OmpA is assembled into a functional protein in vivo and to obtain insight into the of OMP the that we we that the assembly is by the formation of an disulfide between and but only when the cysteine residues in that are in in the folded In contrast, the formation of disulfide bonds between and only the folding of OmpA in for observation is that disulfide bonds that the OmpA β barrel and of the β in by or by other such as the of the β-barrel assembly by a that binds Natl. Acad. Sci. U. S. A. 112: PubMed Scopus Google Scholar). the formation of a disulfide between and OmpA in a circularly form in the of the β to an or the of by chaperones and/or the Bam the circularly form of OmpA in the of and is but the two linked been to be assembled in vivo R. assembly of circularly of the E. coli outer membrane protein Mol. Biol. PubMed Scopus Google Scholar). also of that the assembly of the linked of OmpA required the Bam the of the disulfide bonds not the assembly to Our results the that the assembly of OmpA is in the periplasmic we that the periplasmic is required for the formation of disulfide bonds that greatly OmpA and disulfide observed in a the cysteine that we are all to the cell in the structure of OmpA, it is that catalyzes disulfide the OmpA inserts into the lipid bilayer Bernstein H.D. a for polypeptide across the bacterial outer Cell. 2021; Full Text Full Text PDF PubMed Scopus Google Scholar). to if the OmpA interact in the and are targeted to the OM by chaperones such as or if the fragment with only it binds to its β In either the results that a between the two is required to their a the assembly of OmpA suggests that the two not into the OM and interact to form a β barrel the of OMP β it that the two into a lipid The that only a of cysteine residues disulfide bonds that increase assembly efficiency into the of OmpA to its insertion into the OM. only disulfide bonds the two interact in the and begin of folding into a β The of that required is that the folding of OmpA to be to T.A. of the of the outer membrane protein 112: PubMed Scopus Google Scholar, membrane protein Opin. Struct. Biol. PubMed Scopus Google Scholar). a it is that a β structure to form that and and and to form we if structure is a class of OMPs known as that partially β can form to membrane In it that an segment that the β barrel of a and it to an extracellular to before integration R. Bernstein H.D. of a polypeptide segment into the the assembly of a bacterial Microbiol. 2008; PubMed Scopus Google Scholar). In an of the an OMP that contains a β barrel assembled from also an embedded and assembly intermediates in the R. Peterson J.H. Bernstein H.D. of a bacterial outer membrane protein is in the PubMed Scopus Google Scholar). the embedded segments of OMPs assembly and that are not in β barrel proteins is also to that we if the between the two OmpA that folding is an or an is for that chaperones such as SurA or Skp interact with the two and the formation of bonds that the two the the formation of a folding in the periplasmic In OMP in the of and bacterial for of membrane protein Opin. Struct. Biol. PubMed Scopus Google Scholar, A. Bacterial circularly outer membrane protein Sci. 15: PubMed Scopus Google the that the formation of disulfide bonds between β strands of OmpA assembly be for the of to the of OMP are with models in proteins are targeted to the Bam complex either as partially folded proteins or as unfolded that structure to insertion the barrel and into the membrane by a barrel assembly with an form of In contrast, results not models such as the in newly synthesized OMPs have been to through the lumen in a unfolded and form β that into the OM through a in a N. J.C. Buchanan S.K. The β-barrel assembly machinery in Microbiol. 15: PubMed Scopus Google Scholar). the other not models in the Bam complex the membrane integration of fully folded β barrels by the structure of the lipid it been reported that the to the integration of OMPs into membrane in lipid membrane β-barrel protein folding is by periplasmic lipid and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A.N. Brockwell D.J. Radford S.E. of periplasmic chaperones and membrane protein Mol. Biol. PubMed Scopus Google it that the function of the Bam complex is and that it facilitates OMP assembly by a The E. coli in (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google and To and the from R. Bernstein H.D. of a polypeptide segment into the the assembly of a bacterial Microbiol. 2008; PubMed Scopus Google and the from Silhavy T.J. of the Escherichia coli β-barrel assembly (Bam) is required for to interact with Natl. Acad. Sci. U. S. A. PubMed Scopus Google into by and to as against OmpA extracellular 1 and 4 and an C-terminal have been (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar, Peterson J.H. R. Bernstein H.D. between β barrel assembly and the of Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The in are in and and a circularly permutated OmpA β have been (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar). To the OmpA with and by and as and and in are in The assembled into by assembly J.C. C.A. assembly of to PubMed Scopus Google Scholar). To an into in of the and The fragment OmpA into containing a regulated in PubMed Scopus Google the and To and the of and by E. coli and and and the to into of the the with To the fragment the OmpA by and and to by and with or a in an by 4 with 1 in containing protease and for 20 The with and if of the for the other The folded and unfolded of OmpA by the 1 a (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar). in A of with 4 containing and an A by to a and 5 of the The The by the observed and to the of E. coli with or a in The into an of and to an of to to a of After 5 to the of OmpA for The assembly of OmpA by as and expressed and as (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar). The two OmpA to 20 a of and for formation between the two by as the are the and the contains (13Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.mBio. 2021; 12e00597-21Crossref Scopus (6) Google Scholar). The that they have of with the of and for the by the of the of and and of X. and X. X. X. and The is the of the and not the of the of

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