Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing

Abstract

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...

Evolution of the ssrA degradation tag in Mycoplasma : Specificity switch to a different protease

“…2E). This value is similar to the K m of ClpXP for substrates bearing the E. coli ssrA tag (19,(21)(22)(23). V max for the mf-Lon degradation reaction was surprisingly fast (11.5 Ϯ 0.3 min Ϫ1 Lon 6 Ϫ1 ), approximately three times faster than either ec-Lon or ec-ClpXP degrades unfolded titin (13,19).…”

Intricate Knots in Proteins: Function and Evolution

“…In fact, the first step of protein degradation was shown to be ATP-dependent protein unfolding by threading through a narrow pore (;13 Å in diameter) of a proteasome [21,22]. Such threading into the degradation chamber depends on how easily a protein unfolds, with more stable proteins being released back into solution [23] and unstable ones being degraded. If ATPdependent unfolding proceeds by pulling the C-terminus into a narrow pore [21], then a knot can sterically preclude such translocation, hence preventing protein unfolding and degradation.…”

Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine

“…We tested for an initial lag by determining proteolysis rates for ␤20-or sul20C-tagged substrates containing one to three repeats of titin-I27 CM (19). The reciprocals of these rates, which correspond to ''average'' degradation times, were proportional to substrate length (Fig.…”