The Intrinsically Disordered N-terminal Extension of the ClpS Adaptor Reprograms Its Partner AAA + ClpAP Protease

Torres-Delgado, Amaris; Kotamarthi, Hema Chandra; Sauer, Robert T.; Baker, Tania A.

doi:10.1016/j.jmb.2020.07.007

Cited by 7 publications

(17 citation statements)

References 59 publications

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“…To understand the roles of the different pore loops during ClpS delivery of N-degron substrates, we investigated the requirements for D1 and D2 pore loops during ClpAPS complex formation and degradation of N-end rule substrates. Using our pore-loop variants, we initially tested ClpAPS degradation of J o u r n a l P r e -p r o o f YLFVQELA-GFP, a natively folded N-degron substrate (27,33,34,41). Mutation of three or six D1 or D2 pore loops dramatically reduced the rates of YLFVQELA-GFP degradation compared to those observed with the parental ClpA xl enzyme (Fig.…”

Section: D1 and D2 Pore Loops Are Critical In Clpaps-promoted Degradationmentioning

confidence: 99%

“…For example, residues 20-25, which are required for strong ClpS•ClpA complex formation (33), correspond to an area (teal) on the substrate contacted by three D1 pore loops, consistent with our data that some, but not all, D1 pore loops are required to form this ClpS•ClpA complex. Additionally, residues 17-25 (teal and pink), which represent the region of ClpS required for inhibition of ssrA-degron binding (27,28), are in direct contact with the D1 pore loops. ClpS initially binds to the N-terminal domains of ClpA and ssrA-tagged substrates bind directly in the ClpA channel.…”

Section: Structural Support For D1 and D2 Pore Loop Functions During Clps Deliverymentioning

confidence: 99%

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Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

Zuromski

Kim

Sauer

et al. 2021

Journal of Biological Chemistry

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ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA’s substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to “tug on” and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.

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Section: D1 and D2 Pore Loops Are Critical In Clpaps-promoted Degradationmentioning

confidence: 99%

Section: Structural Support For D1 and D2 Pore Loop Functions During Clps Deliverymentioning

confidence: 99%

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

Zuromski

Kim

Sauer

et al. 2021

Journal of Biological Chemistry

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“…The ClpS adaptor binds and delivers N‐degron substrates for degradation upon association with the ClpAP proteolytic machine 4,38–41 . This ClpS adaptor has been studied extensively in Escherichia coli and some other bacterial systems 4,21,38,42–44 . Structural and biochemical studies of bacterial ClpS revealed how it recognizes type‐2N‐degrons with a preference for Leu and Phe 38–40,42,43,45–47 .…”

Section: Introductionmentioning

confidence: 99%

Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

et al. 2020

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The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.

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“…Adaptors can enhance the action of the protease complex by improving the affinity of the AAA+ protein for the substrate (Wah et al, 2002;Román-Hernández et al, 2011), pulling the substrate to facilitate engagement by the proteolytic complex (Rivera-Rivera et al, 2014), or enabling the assembly of the ATPase hexamers (Kirstein et al, 2006). The presence of adaptors can also reprogram the protease complex by inhibiting the degradation of other protease substrates (Dougan et al, 2002;Torres-Delgado et al, 2020) or preventing autodegradation of the unfoldase (Dougan et al, 2002). Not all proteases and substrates require an adaptor, for instance there are few known examples of proteins activating or reprogramming Lon (Puri and Karzai, 2017) and this protease can exert most of its functions without the aid of accessory proteins.…”

Section: Proteolytic Complexes Based On Clpp and Lonmentioning

confidence: 99%

“…The ClpS N-terminal Extension (NTE) fragment enters the central channel of the protease complex and releases the substrate which is then unfolded and degraded by ClpAP, while ClpS is being recycled (Román-Hernández et al, 2011;Rivera-Rivera et al, 2014). The presence of ClpS significantly reduces the affinity of ClpAP to ssrA-tagged proteins suggesting that it has a complex mode of action, delivering the N-end rule proteins while preventing degradation of other ClpAP substrates (Dougan et al, 2002;Torres-Delgado et al, 2020). No sequelogs of ClpS were identified in Gram-positive bacteria or Archea suggesting that this degradation pathway occurs only in Gram-negative bacteria and eukaryotes (Varshavsky, 2011).…”

Section: N-degron Pathwaymentioning

confidence: 99%

Applications of Bacterial Degrons and Degraders — Toward Targeted Protein Degradation in Bacteria

Izert

Klimecka

Górna

2021

Front. Mol. Biosci.

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A repertoire of proteolysis-targeting signals known as degrons is a necessary component of protein homeostasis in every living cell. In bacteria, degrons can be used in place of chemical genetics approaches to interrogate and control protein function. Here, we provide a comprehensive review of synthetic applications of degrons in targeted proteolysis in bacteria. We describe recent advances ranging from large screens employing tunable degradation systems and orthogonal degrons, to sophisticated tools and sensors for imaging. Based on the success of proteolysis-targeting chimeras as an emerging paradigm in cancer drug discovery, we discuss perspectives on using bacterial degraders for studying protein function and as novel antimicrobials.

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The Intrinsically Disordered N-terminal Extension of the ClpS Adaptor Reprograms Its Partner AAA + ClpAP Protease

Cited by 7 publications

References 59 publications

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

Applications of Bacterial Degrons and Degraders — Toward Targeted Protein Degradation in Bacteria

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