Structure and function of the middle domain of ClpB from Escherichia coli.
ClpB belongs to the Hsp100/Clp ATPase family. Whereas a homologue of ClpB, ClpA, interacts with and stimulates the peptidase ClpP, ClpB does not associate with peptidases and instead cooperates with DnaK/DnaJ/GrpE in an efficient reactivation of severely aggregated proteins. The major difference between ClpA and ClpB is located in the middle sequence region (MD) that is much longer in ClpB than in ClpA and contains several segments of coiled-coil-like heptad repeats. The function of MD is unknown. We purified the isolated MD fragment of ClpB from Escherichia coli (residues 410-570). Circular dichroism (CD) detected a high population of alpha-helical structure in MD. Temperature-induced changes in CD showed that MD is a thermodynamically stable folding domain. Sedimentation equilibrium showed that MD is monomeric in solution. We produced four truncated variants of ClpB with deletions of the following heptad-repeat-containing regions in MD: 417-455, 456-498, 496-530, and 531-569. We found that the removal of each heptad-repeat region within MD strongly inhibited the oligomerization of ClpB, which produced low ATPase activity of the truncated ClpB variants as well as their low chaperone activity in vivo. Only one ClpB variant (Delta417-455) could partially complement the growth defect of the clpB-null E. coli strain at 50 degrees C. Our results show that heptad repeats in MD play an important role in stabilization of the active oligomeric form of ClpB. The heptad repeats are likely involved in stabilization of an intra-MD helical bundle rather than an intersubunit coiled coil.
Kedzierska
Sabina
S
Department of Biochemistry, University of Gdansk, Gdansk, Poland.
Akoev
Vladimir
V
Barnett
Micheal E
ME
Zolkiewski
Michal
M
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D017384:Sequence Deletion; D012996:Solutions; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D012996:Solutions; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C069775:ClpB protein, E coli