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Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria.

Um SH, Kim JS, Song S, Kim NA, Jeong SH, Ha NC - Mol. Cells (2015)

Bottom Line: CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region.We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain.Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agricultural and Life Sciences, Seoul National University, Seoul 151-921, Korea.

ABSTRACT
In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

No MeSH data available.


Related in: MedlinePlus

Sequence and structural comparison. (A) Amino acid sequence alignment of CdDsbA with BdbD (PDB code: 3EU3), SaDsbA (PDB code: 3BCI), and EcDsbA (PDB code: 1A2L). Secondary-structure elements are depicted on the top line with the same color scheme as in Fig. 1. The yellow color indicated the signal peptide. The core DsbA-like domain is shaded in grey. The red box indicates the CXXC motif, and the blue box indicates the loop connecting α5 and α6. The residues preceding the cis-Pro motif are indicated by a red arrow. (B) Structural superimposition of the core DsbA-like domains of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view. The blue box indicates the loop connecting α5 and α6. The CXXC motif is indicated by a red circle. (C) Structural superimposition of the loop region of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view.
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f2-molce-38-8-715: Sequence and structural comparison. (A) Amino acid sequence alignment of CdDsbA with BdbD (PDB code: 3EU3), SaDsbA (PDB code: 3BCI), and EcDsbA (PDB code: 1A2L). Secondary-structure elements are depicted on the top line with the same color scheme as in Fig. 1. The yellow color indicated the signal peptide. The core DsbA-like domain is shaded in grey. The red box indicates the CXXC motif, and the blue box indicates the loop connecting α5 and α6. The residues preceding the cis-Pro motif are indicated by a red arrow. (B) Structural superimposition of the core DsbA-like domains of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view. The blue box indicates the loop connecting α5 and α6. The CXXC motif is indicated by a red circle. (C) Structural superimposition of the loop region of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view.

Mentions: We performed a sequence alignment with the DsbA family proteins, including the structurally and biochemically characterized EcDsbA, BdbD, and SaDsbA proteins (Crow et al., 2009; Heras et al., 2008; Martin et al., 1993) (Fig. 2A). We found that the N-terminal extended region (residues 32–114; colored in cyan) and the C-terminal stretched region (residues 278–289) were unique to CdDsbA. In particular, the N-terminal extended region was unusually long and its interactions with the core DsbA-like domain were not extensive. The homologous and core domain (residues 115–277; Fig. 2A) of the CdDsbA structure was superposed onto other DsbA family proteins, yielding rmsd values for BdbD, SaDsbA, and EcDsbA of 1.2, 2.3, and 4.8 Å, respectively. The structural superposition revealed a notable conformational difference in the loop connecting α-helices α5 and α6 (residues 190–202; Figs. 2A and 2B; blue box). Specifically, the conformations of this connecting loop were divergent among DsbA family proteins (Figs. 2B and 2C). The connecting loop of CdDsbA was located in closest proximity to the CXXC motif (Fig. 2C).


Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria.

Um SH, Kim JS, Song S, Kim NA, Jeong SH, Ha NC - Mol. Cells (2015)

Sequence and structural comparison. (A) Amino acid sequence alignment of CdDsbA with BdbD (PDB code: 3EU3), SaDsbA (PDB code: 3BCI), and EcDsbA (PDB code: 1A2L). Secondary-structure elements are depicted on the top line with the same color scheme as in Fig. 1. The yellow color indicated the signal peptide. The core DsbA-like domain is shaded in grey. The red box indicates the CXXC motif, and the blue box indicates the loop connecting α5 and α6. The residues preceding the cis-Pro motif are indicated by a red arrow. (B) Structural superimposition of the core DsbA-like domains of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view. The blue box indicates the loop connecting α5 and α6. The CXXC motif is indicated by a red circle. (C) Structural superimposition of the loop region of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4546943&req=5

f2-molce-38-8-715: Sequence and structural comparison. (A) Amino acid sequence alignment of CdDsbA with BdbD (PDB code: 3EU3), SaDsbA (PDB code: 3BCI), and EcDsbA (PDB code: 1A2L). Secondary-structure elements are depicted on the top line with the same color scheme as in Fig. 1. The yellow color indicated the signal peptide. The core DsbA-like domain is shaded in grey. The red box indicates the CXXC motif, and the blue box indicates the loop connecting α5 and α6. The residues preceding the cis-Pro motif are indicated by a red arrow. (B) Structural superimposition of the core DsbA-like domains of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view. The blue box indicates the loop connecting α5 and α6. The CXXC motif is indicated by a red circle. (C) Structural superimposition of the loop region of CdDsbA (red), BdbD (blue), SaDsbA (green), and EcDsbA (yellow), displayed in the stereo view.
Mentions: We performed a sequence alignment with the DsbA family proteins, including the structurally and biochemically characterized EcDsbA, BdbD, and SaDsbA proteins (Crow et al., 2009; Heras et al., 2008; Martin et al., 1993) (Fig. 2A). We found that the N-terminal extended region (residues 32–114; colored in cyan) and the C-terminal stretched region (residues 278–289) were unique to CdDsbA. In particular, the N-terminal extended region was unusually long and its interactions with the core DsbA-like domain were not extensive. The homologous and core domain (residues 115–277; Fig. 2A) of the CdDsbA structure was superposed onto other DsbA family proteins, yielding rmsd values for BdbD, SaDsbA, and EcDsbA of 1.2, 2.3, and 4.8 Å, respectively. The structural superposition revealed a notable conformational difference in the loop connecting α-helices α5 and α6 (residues 190–202; Figs. 2A and 2B; blue box). Specifically, the conformations of this connecting loop were divergent among DsbA family proteins (Figs. 2B and 2C). The connecting loop of CdDsbA was located in closest proximity to the CXXC motif (Fig. 2C).

Bottom Line: CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region.We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain.Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agricultural and Life Sciences, Seoul National University, Seoul 151-921, Korea.

ABSTRACT
In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

No MeSH data available.


Related in: MedlinePlus