Specialized AAA+ molecular machines harness the energy of ATP hydrolysis to power protein unfolding and multimer disassembly in all cells1,2
. Such machines, typically active as ring hexamers, initiate unfolding by pulling an exposed peptide tag through a narrow axial pore and ultimately translocate the unfolded polypeptide into the degradation chambers of proteases like ClpXP, ClpAP, HslUV, FtsH, Lon, and the proteasome3–5
(). How, during a power stroke, are nucleotide-dependent changes in the conformation of a AAA+ machine transferred to the polypeptide substrate to drive translocation and unfolding? Answering this question is essential for understanding how these molecular wrecking machines function.
Substrate binding and degradation
In almost all AAA+ unfoldases, a loop with a conserved aromatic-hydrophobic (Ar-Φ) dipeptide protrudes from every subunit into the central pore ()6,7
. Ar-Φ loop mutations have been shown to eliminate or reduce the activity of numerous AAA+ proteases, and it is commonly assumed that these loops play critical roles in translocation and unfolding by transmitting force to substrates7–17
. However, Ar-Φ loops have also been implicated in the initial binding of substrates and in controlling rates of ATP hydrolysis, and defects in either of these processes could also account for the mutant phenotypes observed. An analogy with a macroscopic machine is apt. Imagine trying to determine how an automobile functions by altering various parts. The car can be disabled or severely slowed by obstructing the flow of fuel to the engine, but it would be incorrect to conclude that the fuel pump or carburetor transmits force from the motor to the wheels.
ClpXP is a AAA+ protease that consists of the ClpX ATPase and the ClpP peptidase5
. Here, we characterize the effects of Ar-Φ loop mutations in ClpX from E. coli
. By saturating substrate binding and correcting for altered rates of ATP hydrolysis, we demonstrate specific roles of the Ar-Φ loops in gripping, translocating, and unfolding substrates. Moreover, we show that these loop activities vary as individual ClpX subunits assume ATP-bound, ATP-hydrolyzing, or nucleotide-free states. These results strongly support a model in which nucleotide-dependent conformational changes in the Ar-Φ loops drive substrate translocation and unfolding, with the aromatic ring transmitting force to the polypeptide substrate.