Roles of the ClpX IGF loops in ClpP association, dissociation, and protein degradation

Abstract

IGF-motif loops project from the hexameric ring of ClpX and are required for docking with the self-compartmentalized ClpP peptidase, which consists of heptameric rings stacked back-to-back. Here, we show that ATP or ATPγS support assembly by changing the conformation of the ClpX ring, bringing the IGF loops closer to each other and allowing efficient multivalent contacts with docking clefts on ClpP. In single-chain ClpX pseudohexamers, deletion of one or two IGF loops modestly slows association with ClpP but strongly accelerates dissociation of ClpXP complexes. We probe how changes in the sequence and length of the IGF loops affect ClpX-ClpP interactions and show that deletion of one or two IGF loops slows ATP-dependent proteolysis by ClpXP. We also find that ClpXP degradation is less processive when two IGF loops are deleted.

Keywords: AAA+ protease; ATP-fueled molecular machine; kinetics; protein degradation.

Figure 1

(A) Cartoon of the ClpXP protease degrading a protein substrate. The ClpX hexamer (colored light and dark purple) recognizes a protein substrate (colored green) and uses cycles of ATP hydrolysis to unfold and translocate it into the degradation chamber of the ClpP peptidase (colored dark yellow). The IGF loops of ClpX dock into hydrophobic clefts on ClpP. (B) Top views of the ClpX and ClpP rings, highlighting the six IGF loops of ClpX and seven clefts of ClpP. (C) Sequence‐logo depictions8 of sequence conservation in the IGF loops of ClpX orthologs from γ‐proteobacteria (top) and all bacteria (bottom). The sequence of the E. coli ClpX IGF loop is shown in the middle.

Figure 2

Nucleotide dependence of protease accessibility and relative distance between IGF loops. (A) As assayed by SDS‐PAGE, chymotrypsin cleaved ClpX^ΔN into two major fragments, labeled N and C, which were not observed following chymotrypsin incubation with ClpX^ΔN/ΔIGF or ClpX^ΔN/F270A. Experiments contained ATP (10 mM), chymotrypsin (0.01 mg/mL), and ClpX^ΔN variants (1 μM hexamer). (B) Chymotryptic cleavage of ClpX^ΔN in the presence of different nucleotides. Except for nucleotide identity, experimental conditions were the same as in Panel A. (C) Initial fluorescence of Alexa‐647 labeled T273C ClpX^ΔN in the presence of ATP, ATPγS, ADP, or no nucleotide. The protein concentration was 0.5 μM, and nucleotide was 1.5 mM when present. Values are averages (N = 3) ± SD. (D) Time‐dependent changes in fluorescence of Alexa‐647 labeled T273C ClpX^ΔN under different nucleotide conditions. Other conditions were identical to Panel C. (E) Different concentrations of unlabeled ClpX^ΔN and Alexa‐647 labeled T273C ClpX^ΔN were mixed for 1 h in the absence of nucleotide, 5 mM ADP or ATPγS was added, and fluorescence was measured. (F) Decreased fluorescence caused by increased homo quenching is consistent with the IGF loops being closer together in fluorescent T233C ClpX^ΔN that is bound to ATP compared to ADP. We propose that the IGF loops in ATP‐bound ClpX are properly oriented to make efficient multivalent contacts with the clefts in ClpP, whereas the IGF‐loops in ADP‐bound ClpX can only make a subset of efficient contacts.

Figure 3

Effects of IGF‐loop deletion on ClpP association with ClpX. (A) Normalized BLI association trajectories for experiments performed using 100 nM ClpP. (B) For single‐chain ClpX^ΔN with four IGF loops (loops in subunits AB deleted), k _app varied hyperbolically with ClpP concentration. The line is a non‐linear‐least‐squares fit to the equation k _app = intercept + max•[ClpP]/(K _1/2 + [ClpP]), where k _assn = max/K _1/2. (C) Second‐order rate constants for ClpP association to single‐chain ClpX^ΔN variants with different numbers and configurations of IGF‐loop deletions determined from experiment like those shown in Panels A and B. Values are averages (N = 3) ± SD. All association experiments in this panel contained 2 mM ATP.

Figure 4

Effect of IGF‐loop deletion on dissociation kinetics. (A) Dissociation kinetics for complexes of ClpP and different variants of single‐chain ClpX^ΔN were measured in 1 mM ATP by monitoring changes in BLI response following transfer of the biosensor into buffer lacking ClpP. (B) Half‐lives were calculated from single exponential fits of dissociation experiments like those shown in Panel A. The half‐life for the variant with six IGF loops is a lower limit. Values are averages (N = 3) ± SD.

Figure 5

IGF‐loop deletion affects degradation rates and processivity. (A) Rates of degradation of GFP‐ssrA (20 μM) by ClpP (0.9 μM) and different variants of ClpX (0.3 μM pseudohexamer) were measured by monitoring loss of GFP fluorescence. Values are averages (N = 3) ± SD. (B) Rates of unfolding of a fluorescent Arc‐GCN4‐ssrA (5 μM) by ClpX variants (0.3 μM pseudohexamer) were measured in the presence of 10 mM ATP. (C) Top; cartoon of a substrate containing a TAMRA‐labeled Halo domain, a native titin^I27 domain, three ^V13Ptitin^I27 domains unfolded by carboxymethylation (CM), and a H₆‐ssrA degron. Bottom; SDS‐PAGE assays of the ClpP degradation of this substrate by ClpX^ΔN variants with six, five, or four IGF loops. Note that the variant with four IGF loops shows multiple additional bands between IS and pdp1, indicative of poorly processive degradation. For the enzyme with six IGF‐loops, little accumulation of the pdp1 product or loss of IS occurred after 20 min, probably because the ssrA tag is missing from the majority of substrate molecules because of exopeptidase clipping during purification. Reactions contained substrate (10 μM), ClpP (0.9 μM), single‐chain ClpX^ΔN variants (0.3 μM hexamer equivalents), and ATP (10 mM). Gels were imaged for fluorescence of the TAMRA dye.

Figure 6

Effects of mutations in the IGF loop. (A) Rates of degradation of GFP‐ssrA (20 μM) by ClpP (0.9 μM) and ClpX^ΔN variants containing longer or shorter IGF loops (0.3 μM hexamer). (B) Rates of degradation of GFP‐ssrA (20 μM) by ClpP (0.9 μM) and ClpX^ΔN variants (0.3 μM hexamer) with single‐ or double‐residue substitutions in the IGF loop. (C) Association rate constants determined by BLI experiments using 1 μM ClpP and 2 mM ATP. (D) Dissociation half lives determined by BLI experiments in the presence of 2 mM ATP. In Panels C and D, single‐chain ClpX^ΔN variants had the IGF‐loop deleted from Subunit B and had a wild‐type or mutant IGF motif in Subunit A. In all panels, values are averages (N = 3) ± SD.