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.

Fig. 1.

Degradation of native and unfolded titin_x proteins. (A) Michaelis-Menten plots for ClpXP degradation of unfolded titin substrates. (B) Length dependence of the average time for ClpXP degradation of unfolded titin substrates. (C) Competitive ClpXP degradation of native and unfolded single-domain titin substrates (10 μM each). (D) Lineweaver-Burke analysis of the degradation rate of different concentrations of ³⁵S-labeled CM-titin₁ in the presence of unlabeled titin proteins (1 μM each). Degradation reactions contained 0.1 μM ClpX₆ and 0.3 μM ClpP₁₄.

Fig. 2.

Degradation and properties of FLAG-titin₃. (A) Kinetics of degradation of native ³⁵S-FLAG-titin₃ (0.3 μM) assayed by SDS/PAGE. (B) Degradation of denatured ³⁵S-CM-FLAG-titin₃ (2 μM). (C) Native domains in the titin₁ and titin₃ proteins have similar melting temperatures. (D) Western blots show that partially degraded titin proteins have the FLAG epitope but lack the ssrA tag. Degradation reactions contained 0.3 μM ClpX₆, 0.9 μM ClpP₁₄, and 0.3 μM SspB₂.

Fig. 3.

Partially degraded titin species are released by ClpXP. (A) Production of partially degraded species by ClpXP degradation of different initial concentrations of FLAG-titin₃.(B) SDS/PAGE autoradiogram of ³⁵S-labeled titin proteins and fragments. Lanes 1-3, purified titin₁, titin₂, and titin₃ standards; lanes 4 and 5, purified FLAG-titin₃ degradation products ± subtilisin; lanes 6 and 7, FLAG-titin₃ degradation products after 60 min of ClpXP degradation ± subtilisin. (C) After 4 or 60 min of incubation of 0.3 μM ClpX₆/0.9 μM ClpP₁₄/0.3 μM SspB₂ with (+) or without (-) FLAG-titin₃ (0.3 μM), GFP-ssrA (0.3 μM) was added and its degradation was monitored by loss of fluorescence.

Fig. 4.

Kinetic modeling of ClpXP degradation. (A) Model for ClpXP degradation of native titin₁ and denatured CM-titin_X variants. Units for rate constants are μM^-1·min^-1 for the E + S → ES step and min^-1 for all other steps. For steps where the rate constant for the native and unfolded substrates differs, the native value is in bold and the denatured value is in italics. Denaturation of the native substrate occurs in the ES^* → ET step. The ET → E + P step is unrealistic because product formation and release probably begin as soon as 35 residues of the polypeptide have been translocated and well before ClpX is free to engage another substrate; practically, however, this simplification does not grossly affect the results. (B) Model for degradation of the FLAG-titin₃ substrate. Units are as defined in A. Because denaturation is much slower than translocation, these steps are combined into a single step for the downstream domains. (C) Comparison of experimental values (light gray bars) with simulated values (black bars) calculated by using the model in A.(D) Changes in substrate and partially degraded protein concentrations during ClpXP degradation of FLAG-titin₃. The symbols represent experimental values (see Fig. 2 A). The solid lines were simulated by using the model in B.