Crystal structures of E. coli CcmG and its mutants reveal key roles of the N-terminal beta-sheet and the fingerprint region.
CcmG, also designated DsbE, functions as a periplasmic protein thiol:disulfide oxidoreductase and is required for cytochrome c maturation. Here we report the crystal structures of Escherichia coli CcmG and its two mutants, P144A and the N-terminal fifty seven-residue deletion mutant, and two additional deletion mutants were studied by circular dichroism. Structural comparison of E. coli CcmG with its deletion mutants reveals that the N-terminal beta-sheet is essential for maintaining the folding topology and consequently maintaining the active-site structure of CcmG. Pro144 and Glu145 are key residues of the fingerprint region of CcmG. Pro144 is in cis-configuration, and it makes van der Waals interactions with the active-site disulfide Cys80-Cys83 and forms a C--H...O hydrogen bond with Thr82, helping stabilize the active-site structure. Glu145 forms a salt-bridge and hydrogen-bond network with other residues of the fingerprint region and with Arg158, further stabilizing the active-site structure. The cis-configuration of Pro144 makes the backbone nitrogen and oxygen of Ala143 exposed to solvent, favorable for interacting with binding partners. The key role of cis-Pro144 is verified by the P144A mutant, which contains trans-Ala144 and displays redox property changes. Structural comparison of E. coli CcmG with the recently reported structure of CcmG in complex with the N-terminal domain of DsbD reveals that Tyr141 undergoes conformational changes upon binding DsbD. A cis-proline located at the N-terminus of the first beta-strand of the betabetaalpha motif of the thioredoxin-like domain is a conserved structural feature of the thioredoxin superfamily.
Ouyang
Nan
N
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.
Gao
Yong-Guang
YG
Hu
Hong-Yu
HY
Xia
Zong-Xiang
ZX
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D014443:Tyrosine; C485980:Ccmg protein, E coli; D011490:Protein Disulfide Reductase (Glutathione); D003545:Cysteine