An E35A mutation was introduced to suppress truncation during heterogeneous expression. Oxidized TtCcdA was stable, mono-disperse, and showed promising initial NMR spectra ( Fig. We have screened multiple CcdA proteins from bacteria and archaea for the feasibility of solution NMR studies. Sample conditions and NMR structure determination coli DsbD (EcDsbD), a CcdA homolog, suggests it has an inverted pseudosymmetry 14, 15, which is consistent with the dual accessibilities of the reactive cysteines by the Trx substrates from both sides of the membrane. Cysteine scanning mutagenesis studies probing the solvent accessibility of E. Thus CcdA conducts similar redox reactions on both sides of the membrane. The periplasmic substrates use a Trx-fold domain to react with CcdA 7.
The reducing equivalents are transferred through a cascade of thiol:disulfide exchange reactions from the cytoplasmic general reductant thioredoxin (Trx) to CcdA, across the membrane, then to various periplasmic redox-active proteins ( Fig. A pair of redox active cysteines is located in two conserved PCxxP motifs on TM1 and TM4 12, 13. In addition to bacteria and archeae, CcdA homologs have been found on the thylakoid membrane of chloroplasts to promote cytochrome c maturation, a process essential for photosynthesis 10.ĬcdA has six transmembrane helices arranged in two sequence repeats, TM 1–3 and TM 4–6 ( Supplementary Fig. This is critical for various cellular functions such as cytochrome c biogenesis, lithotrophic sulfur oxidation, oxidative protein folding, and defense against oxidative damage 7– 9.
The membrane electron transporter CcdA (as well as the TM domain of DsbD and ScsB) introduces reducing equivalents from the cytoplasm to the periplasm 1– 6. Crucial reducing pathways exist in the periplasm which rely on external reducing sources. The bacterial cytoplasm is reducing to prevent oxidative damage whereas the periplasm is oxidizing to promote oxidative protein folding. Cells regulate redox homeostasis by compartments.
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