The Escherichia coli ClpA molecular chaperone self-assembles into tetramers.

The Escherichia coli ATP-dependent protease, ClpAP, is composed of the hexameric ATPase/protein-unfoldase, ClpA, and the tetradecameric proteolytic component, ClpP. ClpP proteolytically degrades folded proteins only when associated with the motor protein ClpA or ClpX, both of which use ATP binding and/or hydrolysis to unfold and translocate proteins into the tetradecameric serine protease ClpP. In addition to ClpA's role in regulating the proteolytic activity of ClpP, ClpA catalyzes protein unfolding of proteins that display target sequences to "remodel" them, in vivo, for regulatory roles beyond proteolytic degradation. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA first requires nucleoside triphosphate binding to assemble into an oligomeric form with protein substrate binding activity. In addition to this nucleotide driven assembly activity, ClpA self-associates in the absence of nucleoside triphosphate binding. An examination of the energetics of the nucleotide driven assembly process cannot be performed without a thermodynamic model of the self-assembly process in the absence of nucleotide cofactor. Here we report an examination of the self-association properties of the E. coli ClpA protein unfoldase through the application of analytical ultracentrifugation and light scattering techniques, including sedimentation velocity, sedimentation equilibrium, and dynamic light scattering approaches. In contrast to published results, application of these approaches reveals that ClpA exists in a monomer-tetramer equilibrium (300 mM NaCl, 10 mM MgCl(2), and 25 mM HEPES, pH 7.5 at 25 degrees C). The implications of these results for the E. coli ClpA self-association and ligand linked association activities are discussed.