Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine.

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.

Sculpting the proteome with AAA(+) proteases and disassembly machines.

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.

SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags.

The ssrA-degradation tag sequence contains contiguous binding sites for the SspB adaptor and the ClpX component of the ClpXP protease. Although SspB normally enhances ClpXP degradation of ssrA-tagged substrates, it inhibits proteolysis under conditions that prevent tethering to ClpX. By increasing the spacing between the protease and adaptor-binding determinants in the ssrA tag, substrates were obtained that displayed improved SspB-mediated binding to and degradation by ClpXP. These extended-tag substrates also showed significantly reduced conditional inhibition but bound SspB normally. Both wild-type and mutant tags showed highly dynamic SspB interactions. Together, these results strongly support delivery models in which SspB and ClpX bind concurrently to the ssrA tag, but also suggest that clashes between SspB and ClpX weaken simultaneous binding. During substrate delivery, this signal masking is overcome by tethering SspB to ClpX, which ensures local concentrations high enough to drive tag engagement. This obstruct-then-stimulate mechanism may have evolved to allow additional levels of regulation and could be a common trait of adaptor-mediated protein degradation.

Communication between ClpX and ClpP during substrate processing and degradation.

In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.

Bivalent tethering of SspB to ClpXP is required for efficient substrate delivery: a protein-design study.

SspB homodimers deliver ssrA-tagged substrates to ClpXP for degradation. SspB consists of a substrate binding domain and an unstructured tail with a ClpX binding module (XB). Using computational design, we engineered an SspB heterodimer whose subunits did not form homodimers. Experiments with the designed molecule and variants lacking one or two tails demonstrate that both XB modules are required for strong binding and efficient substrate delivery to ClpXP. Assembly of stable SspB-substrate-ClpX delivery complexes requires the coupling of weak tethering interactions between ClpX and the SspB XB modules as well as interactions between ClpX and the substrate degradation tag. The ClpX hexamer contains three XB binding sites, one per N domain dimer, and thus binds strongly to just one SspB dimer at a time. Because different adaptor proteins use the same tethering sites in ClpX, those which employ bivalent tethering, like SspB, will compete more effectively for substrate delivery to ClpXP.