Unique Contacts Direct High-Priority Recognition of the Tetrameric Mu Transposase-DNA Complex by the AAA+ Unfoldase ClpX

Proc. Natl. Acad. Sci. U.S.A.

2015

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Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNAbinding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation.oxidative damage | proteolysis | degradation signals | Lon

Section: Discussionsupporting

confidence: 89%

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Ahn

Proc. Natl. Acad. Sci. U.S.A.

2015

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“…4). Previous studies showed that ClpXP disassembles the wild-type STC1 with an apparent K M of ∼1.0 μM and an apparent V max of ∼3.1∕ min (21). We found that ClpXP disassembled the double right-end transpososome with an apparent K M of ∼1.1 μM and an apparent V max of ∼3.5∕ min (Fig.…”

Section: Resultssupporting

confidence: 59%

The AAA+ ClpX machine unfolds a keystone subunit to remodel the Mu transpososome

Abdelhakim

Sauer

Proc. Natl. Acad. Sci. U.S.A.

2010

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A hyperstable complex of the tetrameric MuA transposase with recombined DNA must be remodeled to allow subsequent DNA replication. ClpX, a AAAþ enzyme, fulfills this function by unfolding one transpososome subunit. Which MuA subunit is extracted, and how complex destabilization relates to establishment of the correct directionality (left to right) of Mu replication, is not known. Here, using altered-specificity MuA proteins/DNA sites, we demonstrate that transpososome destabilization requires preferential ClpX unfolding of either the catalytic-left or catalytic-right subunits, which make extensive intersubunit contacts in the tetramer. In contrast, ClpX recognizes the other two subunits in the tetramer much less efficiently, and their extraction does not substantially destabilize the complex. Thus, ClpX targets the most stable structural components of the complex. Left-end biased Mu replication is not, however, determined by ClpX's intrinsic subunit preference. The specific targeting of a stabilizing "keystone subunit" within a complex for unfolding is an attractive general mechanism for remodeling by AAAþ enzymes.

“…Because DHFRII is a tetramer, it is likely that a O tag from one subunit tethers the substrate to the N-domain of ClpX, allowing the O tag from a second subunit to be engaged by ClpX for degradation. Recent studies indicate that ClpX disassembly of the tetrameric MuA transposase also involves recognition of multiple classes of sequence elements (29).…”

Section: Discussionmentioning

confidence: 99%

Engineering Synthetic Adaptors and Substrates for Controlled ClpXP Degradation

Davis¹,

Journal of Biological Chemistry

Sauer³

2009

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Facile control of targeted intracellular protein degradation has many potential uses in basic science and biotechnology. One promising approach to this goal is to redesign adaptor proteins, which can regulate proteolytic specificity by tethering substrates to energy-dependent AAA؉ proteases. Using the ClpXP protease, we have probed the minimal biochemical functions required for adaptor function by designing and characterizing variant substrates, adaptors, and ClpX enzymes. We find that substrate tethering mediated by heterologous interaction domains and a small bridging molecule mimics substrate delivery by the wild-type system. These results show that simple tethering is sufficient for synthetic adaptor function. In our engineered system, tethering and proteolysis depend on the presence of the macrolide rapamycin, providing a foundation for engineering highly specific degradation of target proteins in cells. Importantly, this degradation is regulated by a small molecule without the need for new adaptor or enzyme biosynthesis.