Proteolytic machines powered by ATP hydrolysis bind proteins with specific peptide tags, denature these substrates, and translocate them into a sequestered compartment for degradation. To determine how ATP is used during individual reaction steps, we assayed ClpXP degradation of ssrA-tagged titin variants with different stabilities in native and denatured forms. The rate of ATP turnover was 4-fold slower during denaturation than translocation. Importantly, this reduced turnover rate was constant during denaturation of native variants with different stabilities, but total ATP consumption increased with substrate stability, suggesting an iterative application of a uniform, mechanical unfolding force. Destabilization of substrate structure near the degradation tag accelerated degradation and dramatically reduced ATP consumption, revealing an important role for local protein stability in resisting denaturation. The ability to denature more stable proteins simply by using more ATP endows ClpX with a robust unfolding activity required for its biological roles in degradation and complex disassembly.

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal a-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from smallangle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.

Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in all cells. AAA؉ ATPases in these proteases bind protein substrates and power their mechanical denaturation and subsequent translocation into a secluded degradation chamber where polypeptide cleavage occurs. Here, we show that model unfolded substrates are engaged rapidly by ClpXP and are then spooled into the degradation chamber at a rate proportional to their length. Degradation and competition studies indicate that ClpXP initially binds native and unfolded substrates similarly. However, stable native substrates then partition between frequent release and infrequent denaturation, with only the latter step resulting in committed degradation. During degradation of a fusion protein with three tandem native domains, partially degraded species with one and two intact domains accumulated. These processed proteins were not bound to the enzyme, showing that release can occur even after translocation and degradation of a substrate have commenced. The release of stable substrates and committed engagement of denatured or unstable native molecules ensures that ClpXP degrades less stable substrates in a population preferentially. This mechanism prevents trapping of the enzyme in futile degradation attempts and ensures that the energy of ATP hydrolysis is used efficiently for protein degradation.ClpP ͉ ClpX ͉ energy-dependent proteolysis ͉ protein unfolding ͉ titin I27 domain M any cellular processes are powered by molecular machines, which convert energy from ATP into mechanical work. The AAAϩ superfamily of ATPases represent an important class of these machines, functioning in vesicle fusion, cargo transport, DNA and RNA unwinding, remodeling of the cytoskeleton, DNA replication, transposition, and targeted protein degradation (1). In known energy-dependent proteases, hexameric AAAϩ ATPase rings stack against a barrel-shaped peptidase, aligning the central pore of the ATPase with a narrow axial portal of the peptidase. The ATPase ring serves as the control and command center for the proteolytic machine. It binds recognition elements in target proteins, denatures native protein substrates, and translocates the unfolded polypeptide into the proteolytic chamber of the peptidase for degradation (see ref. 2 for review).We have been interested in understanding the coordination of substrate binding, denaturation, and translocation by the ClpXP protease of Escherichia coli (3-6). Most substrates for this ATP-dependent protease have unstructured recognition sequences or degradation tags at their N or C terminus (7). For example, adding an ssrA tag to the C terminus of a protein during tmRNA-mediated ribosome rescue makes it a substrate for ClpXP and other E. coli proteases (8-10). The ssrA tag appears to bind to the central pore of the ClpX 6 ATPase (11), where it serves as a grip or handle that allows the enzyme to apply an unfolding force to the native substrate. Peptide-bond cleavage and product release appear to be fast steps...