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. 2015 Apr 8;10(4):e0123653.
doi: 10.1371/journal.pone.0123653. eCollection 2015.

Domain-swapped Dimer of Pseudomonas Aeruginosa Cytochrome c551: Structural Insights Into Domain Swapping of Cytochrome C Family Proteins

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Free PMC article

Domain-swapped Dimer of Pseudomonas Aeruginosa Cytochrome c551: Structural Insights Into Domain Swapping of Cytochrome C Family Proteins

Satoshi Nagao et al. PLoS One. .
Free PMC article

Abstract

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c551 (PA cyt c551), and Hydrogenobacter thermophilus cytochrome c552 (HT cyt c552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met-heme coordination significantly compared to the monomer. HT cyt c552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structures of horse cyt c and PA cyt c 551.
Horse cyt c (upper left) and PA cyt c 551 (upper right). The hemes and axial ligands are shown as stick models. The heme, the sulfur atoms of the heme axial Met ligand and heme-linked Cys, and the nitrogen atoms of the heme axial His ligand are shown in gray, yellow, and blue, respectively. The secondary structure diagrams of horse cyt c and PA cyt c 551 are depicted at the bottom of the figure. The helices are depicted as arrows in the secondary structure diagrams. The helices and loops are labeled as H1–H4 and L1–L3, respectively.
Fig 2
Fig 2. Crystal structures of monomeric and dimeric WT PA cyt c 551.
(A) Structure of monomeric WT PA cyt c 551 (PDB ID: 351C). (B) Structure of dimeric WT PA cyt c 551 solved in this study (pink and cyan, PDB ID: 3X39). The two protomers are depicted in pink and cyan, respectively. The hemes, Cys12, Cys15, His16, and Met61 are shown as stick models. The N- and C-termini are labeled as N and C, respectively. The hemes and Thr20–Met22 residues (hinge loop) are depicted in dark and pale colors, respectively. The sulfur atoms of the heme axial Met ligand and heme-linked Cys are shown in yellow, and the nitrogen atoms of the heme axial His ligand are shown in blue.
Fig 3
Fig 3. Active site structures of monomeric and dimeric WT PA cyt c 551.
(A) Structure of monomeric WT PA cyt c 551 (PDB ID: 351C). (B) Structure of dimeric WT PA cyt c 551 (PDB ID: 3X39). The heme and side-chains of amino acid residues near the heme (Phe7, Cys12, Ala14, Cys15, His16, Val23, Pro25, Val30, Leu44, Arg47, Ile48, Ser52, Trp56, Pro60, Met61, Pro62, Pro63, Asn64, Leu74, and Val78) are shown as stick models. The sulfur atoms of the heme axial Met ligand and heme-linked Cys are shown in yellow, and the nitrogen atoms of the heme axial His ligand are shown in blue. The cyan strand in the dimeric structure is a region from another molecule. The hemes and Thr20–Met22 residues (hinge loop) are depicted in dark and pale colors, respectively.
Fig 4
Fig 4. CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt c 551.
(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt c 551. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt c 551 shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.
Fig 5
Fig 5. Topology diagrams of PA cyt c 551 and horse cyt c.
(A) Monomeric PA cyt c 551, (B) dimeric PA cyt c 551, (C) monomeric horse cyt c, and (D) dimeric horse cyt c. The helices and loops are labeled as H1–H4 and L1–L3, respectively. The helices are depicted as arrows. The hinge loops in the monomers are depicted in pink.

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This work was partially supported by Grants-in-Aid for Young Scientists (B) (No. 24750163 (S.N.)) and for Scientific Research (B) (No. 26288080 (S.H.)) from JSPS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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