ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation - A novel multi-chaperone system from Escherichia coli

ClpB is a heat-shock protein from Escherichia coil with an unknown function . We studied a possible molecular chaperone activity of ClpB in vitro. Fire fly luciferase was denatured in urea and then diluted into the refolding bu ffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin). Spon taneous reactivation of luciferase was very weak (less than 0.02% of the na tive activity) because of extensive aggregation. Conventional chaperone sys tems (GroEL/GroES and DnaK/ DnaJ/GrpE) or ClpB alone did not reactivate luc iferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activi ty) by suppressing luciferase aggregation. This coordinated function of Clp B and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were a dded to the luciferase refolding solutions after 5-25 min of refolding, Clp B and Dnak/DnaJ/GrpE recovered the luciferase activity from preformed aggre gates. Thus, we have identified a novel multi-chaperone system from E. coli , which is analogous to the Hsp104/Ssal/Ydjl system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat -shock proteins in suppressing and reversing protein aggregation.