The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase.

The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClpP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM. ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.