Self-Association Mechanism of E. coli ClpA Walker B Variants

Abstract

Energy-dependent molecular chaperones are ubiquitous molecular machines involved in protein degradation and remodeling processes essential to cellular vitality. E. coli ClpA is an AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins targeted for degradation into a protease, ClpP. ClpA, like many other AAA+ proteins, assembles into a hexameric ring competent for binding polypeptide substrate clients in the presence of ATP. Each ClpA protomer contains two nucleotide binding domains, NBD1 and NBD2. Hydrodynamic studies have established that ClpA resides in a distribution of oligomers in the absence of nucleotide and addition of nucleotide populates the hexameric state. However, our work shows that a distribution of ClpA oligomers persists in the presence of excess nucleotide, suggesting that macromolecular assembly is thermodynamically linked to nucleotide binding. Lacking a model to quantify the distribution of ClpA oligomeric states, it is currently not possible to predict the concentration of ClpA hexamers available for any given nucleotide and ClpA concentration. In this work, sedimentation velocity studies are used to quantitatively examine the ClpA self-assembly mechanism in the absence and presence of ATP using variants deficient in ATP hydrolysis at either one or both NBDs. The stoichiometry and affinity of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on nucleotide concentration. This analysis is the first step in a detailed quantitative understanding of how the twelve nucleotide binding and hydrolysis sites within the hexametric ring coordinate ATP hydrolysis and coupling to polypeptide translocation.