Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX

Abstract

The hexameric AAA+ ring of Escherichia. coli ClpX, an ATP-dependent protein unfolding and translocation machine, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+ ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP-hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U⇔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding, and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+ ring activity.

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

“…However, we do not observe any activation of hydrolytic rate at micromolar ADP concentrations (Fig. 2c), although such an effect was recently reported for the distant homolog ClpX (60). In ClpX, ϳ100 M ADP is thought to set the homo-oligomeric ring into a catalytically activated conformational state, as evidenced by a moderate jump in ATPase activity.…”

Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle

“…This decrease in the association rate in the presence of excess ADP probably reflects reduced ATP binding, as a result of competitive inhibition, and increased ADP occupancy of ClpX subunits. 8 …”

Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine

“…A 2-nm step requires a kinetic burst of two power strokes, etc. Concerted p1-loop movement during a single power stroke does not require concerted or sequential ATP hydrolysis in the ClpX ring, and multiple experiments support probabilistic ATP hydrolysis by individual subunits (Martin et al, 2005; Stinson et al, 2015). Therefore, each stochastic hydrolysis event appears to result in a 1-nm power stroke in which all of the p1-loops move.…”