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EcoSal Plus

Domain 2: Cell Architecture and Growth

Disulfide Bond Formation in the Periplasm of

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  • Authors: Bruno Manta1, Dana Boyd2, and Mehmet Berkmen3
  • Editors: James M. Slauch4, Michael Ehrmann5
    Affiliations: 1: New England Biolabs, Ipswich, MA 01938; 2: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115; 3: New England Biolabs, Ipswich, MA 01938; 4: The School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL; 5: Center for Medical Biotechnology (ZMB), University of Duisburg-Essen, Essen, Germany
  • Received 15 November 2018 Accepted 02 January 2019 Published 13 February 2019
  • Address correspondence to Mehmet Berkmen, [email protected]
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  • Abstract:

    The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that disulfide bond formation is catalyzed by enzymes, the field of oxidative folding of proteins was born. played a central role as a model organism for the elucidation of the disulfide bond-forming machinery. Since then, many of the enzymatic players and their mechanisms of forming, breaking, and shuffling disulfide bonds have become understood in greater detail. This article summarizes the discoveries of the past 3 decades, focusing on disulfide bond formation in the periplasm of the model prokaryotic host .

  • Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018


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The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that disulfide bond formation is catalyzed by enzymes, the field of oxidative folding of proteins was born. played a central role as a model organism for the elucidation of the disulfide bond-forming machinery. Since then, many of the enzymatic players and their mechanisms of forming, breaking, and shuffling disulfide bonds have become understood in greater detail. This article summarizes the discoveries of the past 3 decades, focusing on disulfide bond formation in the periplasm of the model prokaryotic host .

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Figure 1

A protein requiring disulfide bonds for its stability is translocated into the periplasm via the SecYEG translocon with its cysteines (arbitrarily labeled C1 to C4) in a reduced state (substrate) . Disulfide bond formation is catalyzed by DsbA, either during translocation, after translocation, or both . DsbA is reoxidized back to its active oxidized state by DsbB, and DsbB is oxidized by ubiquinone in aerobic conditions or by menaquinone in anaerobic conditions . If the substrate is misoxidized (substrate), its disulfide bonds are isomerized to their native oxidized states (substrate) by DsbC . DsbC along with DsbG and CcmG are maintained in their active reduced states by DsbD . DsbD in turn is reduced by the cytoplasmic thioredoxin TrxA, which receives its reducing potential ultimately from cytoplasmic pools of NADPH . CcmG maintains CcmH in a reduced state. Through the interaction of CcmH with the CcmCDEF membrane complex, oxidized cytochrome-c is reduced, enabling it to form thioether covalent bonds with its heme cofactor . Proteins with thioredoxin folds are in red, and cysteines are in yellow. The amino acid residue numbers of the redox-active cysteines are indicated.

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 2

Crystal structure of oxidized DsbA (PDB accession number 1FVK). The thioredoxin domain is in blue, and the α-helical domain is in red. The active site disulfide bond and the critical proline are indicated.

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 3

DsbA catalyzes the formation of disulfide bonds in a polypeptide with reduced cysteines. The cysteines within the Cys-X-X-Cys active site of DsbA are oxidized (S-S), and the thiol side-groups of cysteine residues in the substrate are reduced (SH) (panel 1). Disulfide bond formation is initiated by deprotonation of a thiol group in the substrate (panel 2). The resulting thiolate anion can initiate a nucleophilic attack on the disulfide bond of DsbA (panel 3). The resolution of the mixed-disulfide bonded complex could occur by deprotonation of another thiol group (panel 4), which can attack the substrate-DsbA disulfide bond (panel 5). The result of this reaction is the oxidation of the substrate and the reduction of DsbA (panel 6).

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 4

The topology of DsbB based on alkaline phosphatase fusion studies ( 188 ). The active site cysteines are shown in their oxidized states, and the putative transmembrane domain amino acids are highlighted.

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 5

A reduced DsbA interacts with oxidized DsbB, resulting in the reoxidation of DsbA and reduction of DsbB. The DsbA-DsbB complex is formed via a disulfide bond between C of DsbA and C of the second periplasmic loop of DsbB. The resolution of this complex is believed to occur through two pathways. In pathway A, a disulfide bond is formed between the first and second periplasmic loop, which is resolved by the oxidation of DsbB by quinones. In pathway B, the DsbA-DsbB complex is resolved by quinones without the interaction of the first periplasmic loop. Figure based on Fig. 8 in reference 84 .

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 6

The crystal structure of the homodimer DsbC showing the two domains (thioredoxin in blue and dimerization in green) separated by the short α-helix linker in red (PDB accession number 1EEJ). Redox-active cysteines (C to C) are represented as yellow spheres, and the structural disulfide bond (C to C) is indicated. The molecular surface of DsbC is superimposed, visualizing the pocket formed by the dimerization of DsbC. Top-down view of DsbC displaying the noncharged pocket devoid of acidic (red) and basic (blue) amino acid residues.

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 7

For clarity, only a monomer of DsbC is shown. Reduced active DsbC recognizes misoxidized substrate and forms a mixed-disulfide bonded complex. This complex can be resolved by the reduction of the disulfide bond in the substrate, resulting in the oxidation of DsbC . A secondary cycle of reduction is necessary for the substrate to be fully reduced , allowing DsbA to reoxidize the substrate . Alternatively, the disulfide bonds in the complex can be shuffled by the isomerase action of DsbC, resulting in native disulfide bonded substrate and reduced DsbC .

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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Figure 8

The predicted membrane topology of DsbD from the web-based program Phobius (http://phobius.binf.ku.dk). The immunoglobin-like α domain is crystallized (PDB accession number 1L6P) devoid of its signal peptide from the amino acids Arg to Asn. The amino acids of the β domain from Asn to Thr are depicted as circles. The redox-active cysteines (C to C) are highlighted as yellow circles. The thioredoxin-like γ domain from Ala to Pro is crystallized (PDB accession number 1UC7). Active site cysteines in the crystal structures are shown as yellow spheres (α domain C to C and β domain C to C). The membrane is shaded gray.

Citation: Manta B, Boyd D, Berkmen M. 2019. Disulfide Bond Formation in the Periplasm of , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0012-2018
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