A novel ClpX degron targets the E. coliantitoxin MqsA for degradation by ClpXP

• Published in: The FASEB journal Vol. 36 Suppl 1
• Main Authors: Vos, Margaret R, Piraino, Benjamin, LaBreck, Christopher J, Rahmani, Negar, Trebino, Catherine E, Peti, Wolfgang, Camberg, Jodi L, Page, Rebecca
• Format: Journal Article
• Language: English
• Published: United States 01.05.2022
• ISSN: 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R2150

It is well established that the antitoxins of toxin-antitoxin (TA) systems are selectively degraded by bacterial proteases in response to stress. However, how distinct stressors result in the selective degradation of specific antitoxins remains unanswered. MqsRA is a TA system activated by stress, including oxidation. To understand how, at a molecular level, MqsA is targeted for degradation by bacterial proteases and to advance our understanding of how substrates are recognized by bacterial proteolytic systems, especially ClpXP, we studied the mechanism of MqsA recognition and degradation by the ClpXP protease. Here, we show that MqsA is efficiently degraded by ClpXP in the absence of zinc, and that both zinc and MqsR binding antagonize ClpXP mediated degradation. Further, using biomolecular NMR based chemical shift perturbation experiments, we show that folded, metal-bound MqsA does not interact with the ClpX N-domain (a small recognition domain that binds a subset of ClpXP substrates and adaptor proteins), but instead ClpX interacts only with unfolded, metal-free MqsA. This led to the identification of a cryptic N-domain recognition sequence in unfolded MqsA that targets it for ClpXP degradation. Furthermore, we discovered this sequence is transplantable and sufficient to target a fusion protein for in vivo and in vitro degradation. This recognition sequence defines a novel ClpX degron, which differs in sequence from all previously described degrons. Finally, we present a model in which oxidative stress causes rapid cysteine oxidation of nascent, zinc free MqsA, and exposure of the degron for ClpXP mediated degradation.