Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli

The heat-shock protein ClpB is a protein-activated ATPase that is essential for survival of Escherichia coli at high temperatures. ClpB has also recen tly been suggested to function as a chaperone in reactivation of aggregated proteins. In addition, the clpB gene has been shown to contain two transla tional initiation sites and therefore encode two polypeptides of different size. To determine the structural organization of ClpB, the ClpB proteins w ere subjected to chemical cross-linking analysis and electron microscopy. T he average images of the ClpB proteins with end on orientation revealed a s even-membered, ring-shaped structure with a central cavity. Their side-on v iew showed a two-layered structure with an equal distribution of mass acros s the equatorial plane of the complex. Since the ClpB subunit has two large regions containing consensus sequences for nucleotide binding, each layer of the ClpB heptamer appears to represent the side projection of one of the major domains arranged on a ring. Ln the absence of salt and ATP, the ClpB proteins showed a high tendency to form a heptamer. However, they dissocia ted into various species of oligomers with smaller sizes, depending on salt concentration. Above 0.2 M NaCl, the ClpB proteins behaved most likely as a monomer in the absence of ATP, but assembled into a heptamer in its prese nce. Furthermore, mutations of the first ATP-binding site, but not the seco nd site, prevented the ATP-dependent oligomerization of the ClpB proteins i n the presence of 0.3 M NaCl. These results indicate that ClpB has a heptam eric ring-shaped structure with a central cavity and this structural organi zation requires Am binding to the first nucleotide-binding site localized t o the N-terminal half of the ATPase. (C) 2000 Academic Press.