AAA+ Molecular Machines: Firing on All Cylinders
Abstract
The AAA+ ATPase motif is found in proteins from all kingdoms of life. The cellular roles of these proteins are diverse; hence the AAA+ designation for ATPase’s associated with various cellular activities. Many of these enzymes act as machines, using energy generated from the binding and hydrolysis of ATP to remodel a variety of macromolecular assemblies in the cell [1]. For example, AAA+ subunits of the proteosome in eukaryotes and the Clp protease in prokaryotes denature stably folded proteins [2,3]. Other members of the family disassemble oligomeric protein structures, like NSF, which takes apart membrane fusion complexes [4]. In addition, some AAA+ enzymes, such as the RuvB DNA helicase, act on nucleic acids instead of proteins [5]. Structural and biochemical studies of AAA+ enzymes have shown that they often assemble into oligomeric structures and share a high degree of structural similarity, particularly in the ATP binding domain [6]. Although several models have been proposed for how these molecular machines work, the mechanism is still not well understood. Martin et al. [7] have used a novel approach to identify the fundamental, mechanical principles that underlie the bacterial ClpX AAA+ ATPase motor. Their approach can be used with other AAA+ protein machines to determine whether these operating principles are shared by additional members of this diverse protein family. This technique is also applicable to other oligomeric proteins, not just those in the AAA+ family, providing a powerful tool to investigate the function of many protein complexes. The ClpX AAA+ protein from Escherichia coli forms a barrelshaped oligomer made of six identical subunits [3]. It can act alone as a chaperone or as a protease when assembled with another barrel-shaped oligomeric protein, ClpP. ClpX binds to folded substrates that have specific recognition sequences [8]. It then uses energy derived from ATP hydrolysis to unfold and translocate substrate proteins through the central pore of the hexamer [9]. Many rounds of ATP hydrolysis are needed to fully unfold and translocate a substrate protein [10]. When assembled with the ClpP protease, ClpX delivers the unfolded substrate protein directly into the central cavity of ClpP for degradation (Figure 1) [3]. Structural and biochemical studies of AAA+ ATPase proteins have generated two basic models for how these motors function [1]. In some structures, nucleotides are bound to all subunits, suggesting that the motor functions via a concerted mechanism in which all subunits hydrolyze ATP simultaneously [11,12] (Figure 2). In contrast, other structures have nucleotides bound to only a few of the subunits [12,13]. This asymmetry in nucleotide binding has led to models proposing a sequential mechanism for motor function. According to these models, the enzyme functions like a rotary engine: individual subunits hydrolyze ATP asynchronously and ATP hydrolysis proceeds in a predetermined, sequential order around the AAA+ ATPase ring (Figure 2). A key experiment to test whether AAA+ motors use either a concerted or sequential mechanism would be to form mixed oligomers of active and inactive subunits, varying both their number and placement in AAA+ ATPase protein machines use the power obtained from ATP hydrolysis to remodel macromolecular assemblies in the cell. Recent work in Escherichia coli on the ClpX AAA+ protein reveals fundamental mechanical principles that underlie ClpX motor function.