Contributions of cysteine residues in Zn2 to zinc fingers and thiol-disulfide oxidoreductase activities of chaperone DnaJ.
Escherichia coli DnaJ, possessing both chaperone and thiol-disulfide oxidoreductase activities, is a homodimeric Hsp40 protein. Each subunit contains four copies of a sequence of -CXXCXGXG-, which coordinate with two Zn(II) ions to form an unusual topology of two C4-type zinc fingers, C144DVC147Zn(II)C197NKC200 (Zn1) and C161PTC164Zn(II)C183PHC186 (Zn2). Studies on five DnaJ mutants with Cys in Zn2 replaced by His or Ser (C183H, C186H, C161H/C183H, C164H/183H, and C161S/C164S) reveal that substitutions of one or two Cys residues by His or Ser have little effect on the general conformation and association property of the molecule. Replacement of two Cys residues by His does not interfere with the zinc coordination. However, replacement of two Cys by Ser results in a significant decrease in the proportion of coordinated Zn(II), although the unique zinc finger topology is retained. The mutants of C183H, C186H, and C161S/C164S display full disulfide reductase activity of wild-type DnaJ, while C161H/C183H and C164H/183H exhibit severe defect in the activity. All of the mutations do not substantially affect the chaperone activity. The results indicate that the motif of -CXXC- is critical to form an active site and indispensable to the thiol-disulfide oxidoreductase activity of DnaJ. Each -CXXC- motif in Zn2 but not in Zn1 functions as an active site.
Shi
Yuan-yuan
YY
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Tang
Wei
W
Hao
Shu-feng
SF
Wang
Chih-Chen
CC
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012694:Serine / Q000235:genetics; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers / Q000235:genetics
C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D012694:Serine; D006639:Histidine; D011490:Protein Disulfide Reductase (Glutathione); D015032:Zinc; D003545:Cysteine