Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone.

The ClpX heat shock protein of Escherichia coli is a member of the universally conserved Hsp100 family of proteins, and possesses a putative zinc finger motif of the C(4) type. The ClpX is an ATPase which functions both as a substrate specificity component of the ClpXP protease and as a molecular chaperone. Using an improved purification procedure we show that the ClpX protein is a metalloprotein complexed with Zn(II) cations. Contrary to other Hsp100 family members, ClpXZn(II) exists in an oligomeric form even in the absence of ATP. We show that the single ATP-binding site of ClpX is required for a variety of tasks, namely, the stabilization of the ClpXZn(II) oligomeric structure, binding to ClpP, and the ClpXP-dependent proteolysis of the lambdaO replication protein. Release of Zn(II) from ClpX protein affects the ability of ClpX to bind ATP. ClpX, free of Zn(II), cannot oligomerize, bind to ClpP, or participate in ClpXP-dependent proteolysis. We also show that ClpXDeltaCys, a mutant protein whose four cysteine residues at the putative zinc finger motif have been replaced by serine, behaves in similar fashion as wild type ClpX protein whose Zn(II) has been released either by denaturation and renaturation, or chemically by p-hydroxymercuriphenylsulfonic acid.