Structural and Functional Studies of the E. Coli ClpA Molecular Motor

Abstract

ATP dependent proteases, such as the E. coli ClpAP and eukaryotic 26 S proteasome are critical components of protein quality control pathways. These proteases have the responsibility of removing misfolded proteins that can occur during heat shock or stress. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and either the hexameric ClpA (84.2 kDa monomer) or ClpX (46.2 kDa monomer) ATPase/protein unfoldase. In addition to its proteolytic activity, ClpA has protein remodeling activity and therefore, in the absence of ClpP, is considered a molecular chaperone. From sequence analysis, ClpA has been found to be a member of the ATPases Associated with various Activities (AAA+) family of proteins. This family of proteins includes a number of oligomeric chaperones and some DNA helicases. Here we report a rigorous investigation of the self association properties of the E. coli ClpA chaperone, including the ligand linked assembly of the structure active in polypeptide translocation. This has been done by employing sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments. We also employ rapid mixing kinetic approaches to examine the polypeptide translocation activities of ClpA and ClpAP. Thus far we have shown that ClpA translocates from the carboxy- to amino-terminus without dissociating under single-turnover conditions and thus we consider ClpA to be a processive and directional polypeptide translocase. In addition, we will discuss the first determination of a kinetic step-size for a polypeptide translocase, where the kinetic step-size is defined as the number of amino acids translocated per repeating step.