![]() We also characterize subsequent unfolding/translocation steps that initiate processive degradation. Here, we establish the molecular basis of the recognition step in targeted ClpXP degradation of ssrA-tagged proteins. ![]() Subsequent mechanical reactions requiring ATP hydrolysis unfold adjacent regions of native protein structure and then translocate the denatured polypeptide through the channel and into the degradation chamber of the double-ring ClpP 14 peptidase for proteolysis ( Sauer and Baker, 2011 Olivares et al., 2018). The ssrA tag initially binds in the axial channel of the hexameric AAA+ ClpX ring, where the pore-1, pore-2, and RKH loops contribute to recognition ( Siddiqui et al., 2004 Farrell et al., 2007 Martin et al., 2008b Iosefson et al., 2015). The terminal Ala-Ala- coo – dipeptide of this degron is the most important element for ClpXP degradation ( Flynn et al., 2001), and related degrons ending in Ala-Ala target other cellular proteins to ClpXP ( Flynn et al., 2003 Neher et al., 2006 Lytvynenko et al., 2019). coli ssrA tag is AANDENYALAA- coo – ( Keiler et al., 1996). A different bacterial mechanism, which is similar to eukaryotic systems, adds alanine tails to the nascent polypeptide during the ribosome-rescue reaction ( Buskirk and Green, 2017 Lytvynenko et al., 2019). During rescue, tmRNA binds in the empty A-site of a stalled ribosome, adds a charged alanine to the nascent polypeptide in a tRNA-like reaction, replaces the original mRNA with a short open reading frame that directs translation of the remaining residues of the ssrA degron, and finally recruits translation termination factors via a stop codon. ![]() Escherichia coli ClpXP, for example, degrades proteins bearing a C-terminal degron called the ssrA tag that is added during tmRNA-rescue of stalled ribosomes ( Keiler et al., 1996 Keiler, 2015). In bacteria and eukaryotic organelles, AAA+ proteases typically recognize substrates via short N- or C-terminal peptide sequences. ClpX (a AAA+ protein unfoldase/translocase) and/or ClpP (a self-compartmentalized peptidase) are also potential therapeutic targets for bacterial pathogens, such as Mycobacterium tuberculosis, as well as in human developmental defects, hematological disease, and cancer ( Bhandari et al., 2018). IntroductionĬlpXP and related AAA+ proteases maintain cellular health by degrading incomplete, damaged, or unneeded proteins in a process that must be specific to avoid destruction of essential intracellular proteins ( Sauer and Baker, 2011). Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. ![]() In Escherichia coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity.
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