Abstract
When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. 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. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. 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. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.
Highlights
ClpXP 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)
The ssrA degron bound in the top of the ClpX channel in a structure we call the recognition complex and moved ~25 Å or 6 residues deeper into the channel in a structure we call the intermediate complex (Figure 1B)
Our cryo-EM structures of the recognition and intermediate complexes represent the first instances in which ClpX interactions with specific side chains of a protein substrate have been visualized
Summary
ClpXP 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). 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). 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 recruits translation termination factors via a stop codon. Subsequent mechanical reactions requiring ATP hydrolysis unfold adjacent regions of native protein structure and translocate the denatured polypeptide through the channel and into the degradation chamber of the double-ring ClpP14 peptidase for proteolysis (Sauer and Baker; 2011; Olivares et al, 2018). We characterize subsequent unfolding/translocation steps that initiate processive degradation
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