Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation.

The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.

Roles of double-loop (130~159 aa and 175~209 aa) in ClpY(HslU)-I domain for SulA substrate degradation by ClpYQ(HslUV) protease in Escherichia coli.

An Escherichia coli ATP-dependent two-component protease, ClpYQ(HslUV), targets the SulA molecule, an SOS induced protein. ClpY recognizes, unfolds and translocates the substrates into the proteolytic site of ClpQ for degradation. ClpY is divided into three domains N, I and C. The N domain is an ATPase; the C domain allows for oligomerization, while the I domain coordinates substrate binding. In the ClpYQ complex, two layer pore sites, pore I and II, are in the center of its hexameric rings. However, the actual roles of two outer-loop (130~159 aa, L1 and 175~209 aa, L2) of the ClpY-I domain for the degradation of SulA are unclear. In this study, with ATP, the MBP-SulA molecule was bound to ClpY oligomer(s). ClpYΔL1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpYΔL2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpYΔpore I had a lower SulA-binding activity. ClpYΔ pore I ΔL2 showed the lowest SulA-binding activity. In addition, ClpY (Q198L, Q200L), with a double point mutation in loop 2, formed stable oligomers. It also had a subtle increase in SulA-binding activity, but displayed less proteolytic activity. As a result, loop 2 has an effect on ClpY oligomerization, substrate binding and delivery. Loop 1 has a role as a gate, to prevent excessive substrate binding. Thus, accordingly, ClpY permits the formation of SulA-ClpY(6x), with ATP(s), and this complex then docks through ClpQ(6x) for ultimate proteolytic degradation.

A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis.

The I domain of HslU sits above the AAA+ ring and forms a funnel-like entry to the axial pore, where protein substrates are engaged, unfolded, and translocated into HslV for degradation. The L199Q I-domain substitution, which was originally reported as a loss-of-function mutation, resides in a segment that appears to adopt multiple conformations as electron density is not observed in HslU and HslUV crystal structures. The L199Q sequence change does not alter the structure of the AAA+ ring or its interactions with HslV but increases I-domain susceptibility to limited endoproteolysis. Notably, the L199Q mutation increases the rate of ATP hydrolysis substantially, results in slower degradation of some proteins but faster degradation of other substrates, and markedly changes the preference of HslUV for initiating degradation at the N or C terminus of model substrates. Thus, a structurally dynamic region of the I domain plays a key role in controlling protein degradation by HslUV.

The degradation of RcsA by ClpYQ(HslUV) protease in Escherichia coli.

In Escherichia coli, RcsA, a positive activator for transcription of cps (capsular polysaccharide synthesis) genes, is degraded by the Lon protease. In lon mutant, the accumulation of RcsA leads to overexpression of capsular polysaccharide. In a previous study, overproduction of ClpYQ(HslUV) protease represses the expression of cpsB∷lacZ, but there has been no direct observation demonstrating that ClpYQ degrades RcsA. By means of a MBP-RcsA fusion protein, we showed that RcsA activated cpsB∷lacZ expression and could be rapidly degraded by Lon protease in SG22622 (lon(+)). Subsequently, the comparative half-life experiments performed in the bacterial strains SG22623 (lon) and AC3112 (lon clpY clpQ) indicated that the RcsA turnover rate in AC3112 was relatively slow and RcsA was stable at 30°C or 41°C. In addition, ClpY could interact with RscA in an in vitro pull-down assay, and the more rapid degradation of RcsA was observed in the presence of ClpYQ protease at 41°C. Thus, we conclude that RcsA is indeed proteolized by ClpYQ protease.

Regulation of clpQ⁺Y⁺ (hslV⁺U⁺) gene expression in Escherichia coli.

The Escherichia coli ClpYQ (HslUV) complex is an ATP-dependent protease, and the clpQ⁺Y⁺ (hslV⁺U⁺) operon encodes two heat shock proteins, ClpQ and ClpY, respectively. The transcriptional (op) or translational (pr) clpQ⁺::lacZ fusion gene was constructed, with the clpQ⁺Y⁺ promoter fused to a lacZ reporter gene. The clpQ⁺::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λlpQ⁺::lacZ⁺ (op), a transcriptional fusion gene, was used to form lysogens in the wild-type, rpoH or/and rpoS mutants. Upon shifting the temperature up from 30 ° C to 42 ° C, the wild-type λclpQ⁺::lacZ⁺ (op) demonstrates an increased ß-galactosidase (ßGal) activity. However, the ßGal activity of clpQ⁺::lacZ⁺ (op) was decreased in the rpoH and rpoH rpoS mutants but not in the rpoS mutant. The levels of clpQ⁺::lacZ⁺ mRNA transcripts correlated well to their ßGal activity. Similarly, the expression of the clpQ⁺::lacZ⁺ gene fusion was nearly identical to the clpQ⁺Y⁺ transcript under the in vivo condition. The clpQ(m1)::lacZ⁺, containing a point mutation in the -10 promoter region for RpoH binding, showed decreased ßGal activity, independent of activation by RpoH. We conclude that RpoH itself regulates clpQ⁺Y⁺ gene expression. In addition, the clpQ⁺Y⁺ message carries a conserved 71 bp at the 5' untranslated region (5'UTR) that is predicted to form the stem-loop structure by analysis of its RNA secondary structure. The clpQ(m2)Δ40::lacZ⁺, with a 40 bp deletion in the 5'UTR, showed a decreased ßGal activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ⁺Y⁺ message.