Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine

Abstract

ATP-powered proteases enforce protein quality-control and regulation in all domains of life. ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. X-ray crystal structures show that some ClpX subunits occupy nucleotide-loadable conformations and others occupy unloadable conformations. Furthermore, biochemical evidence suggests that multiple classes of nucleotide-loadable subunits exist. How asymmetry among subunits is coordinated to achieve mechanical function has remained unclear. Using a combination of mutagenesis, disulfide crosslinking, and fluorescence methods to assay the conformations and nucleotide-binding properties of individual subunits, we demonstrate dynamic interconversion of loadable and unloadable subunits. Such interconversion is required to couple ATP hydrolysis by ClpX to mechanical work, plays a role in substrate binding and ClpP interaction, and is not strictly coupled to the ATP hydrolysis cycle. ATP binding to different classes of subunits drives allosteric changes in ring conformation to allow hydrolysis and coupled machine function, and we present a subunit-specific single molecule nucleotide occupancy assay to elucidate details of this process.