Molecular Mechanisms of Enzyme Catalyzed Protein Unfolding and Translocation by Class 1 AAA+ Motor

Abstract

AAA+ molecular motors involved in protein quality control are at the heart of many biological functions. Here I will discuss our efforts on three model AAA+ driven motors, the prokaryotic ClpA and ClpB and eukaryotic Hsp104. ClpA is a hexameric ring motor that uses the energy from ATP binding/hydrolysis to processively translocate a polypeptide substrate through its axial channel for either protein remodeling or ATP dependent proteolysis. ClpA associates with the tetradecameric serine protease, ClpP, to form the ATP dependent protease ClpAP. In contrast, ClpB and Hsp104 have the unique ability to disrupt protein aggregates in vivo and do not associate with any known proteases. Due to the structural similarity between ClpA and ClpB/Hsp104 it has long been hypothesized that ClpB and Hsp104 processively translocate a polypeptide through their axial channels of the hexameric ring structures. However, for ClpB/Hsp104 and related enzymes, this hypothesis has been difficult to test because these enzymes do not covalently modify the substrate on which they translocate. We have developed a transient state kinetics strategy that is sensitive to processive translocation catalyzed by ClpA in the absence of proteolytic degradation. Using this approach, we have shown that ClpA employs a different molecular mechanism when translocating polypeptide in the absence of the protease vs. when it is present. Further, we have shown that ClpB and Hsp104 take, at most, two translocation steps on the polypeptide substrate before rapid dissociation. These results reveal that ClpB and Hsp104 exhibit low processivity, inconsistent with the complete threading model. These findings and the application of these approaches have broad impact on the examination of many AAA+ molecular motors that do not covalently modify the substrate on which they operate.