The C-terminal (331-376) sequence of Escherichia coli DnaJ is essential for dimerization and chaperone activity: a small angle X-ray scattering study in solution.
DnaJ, an Escherichia coli Hsp40 protein composed of 376 amino acid residues, is a chaperone with thioldisulfide oxidoreductase activity. We present here for the first time a small angle x-ray scattering study of intact DnaJ and a truncated version, DnaJ (1-330), in solution. The molecular weight of DnaJ and DnaJ (1-330) determined by both small angle x-ray scattering and size-exclusion chromatography provide direct evidence that DnaJ is a homodimer and DnaJ (1-330) is a monomer. The restored models show that DnaJ is a distorted omega-shaped dimeric molecule with the C terminus of each subunit forming the central part of the omega, whereas DnaJ (1-330) exists as a monomer. This indicates that the deletion of the C-terminal 46 residues of DnaJ impairs the association sites, although it does not cause significant conformational changes. Biochemical studies reveal that DnaJ (1-330), while fully retaining its thiol-disulfide oxidoreductase activity, is structurally less stable, and its peptide binding capacity is severely impaired relative to that of the intact molecule. Together, our results reveal that the C-terminal (331-376) residues are directly involved in dimerization, and the dimeric structure of DnaJ is necessary for its chaperone activity but not required for the thiol-disulfide oxidoreductase activity.
Shi
Yuan-yuan
YY
National Laboratory of Biomacromolecules, Institute of Biophysics, Beijing, China.
Hong
Xin-guo
XG
Wang
Chih-chen
CC
D002845:Chromatography; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015233:Models, Statistical; D018832:Molecular Chaperones / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014965:X-Rays; D016335:Zinc Fingers
D004220:Disulfides; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D013438:Sulfhydryl Compounds; D010088:Oxidoreductases