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.
Subject(s)
Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , RNA Folding , RNA-Binding Proteins/metabolism , Cryoelectron Microscopy , Endopeptidase Clp/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Protein Conformation , RNA-Binding Proteins/chemistry , Ribosomes/metabolismABSTRACT
In the AAA+ ClpXP protease, ClpX uses repeated cycles of ATP hydrolysis to pull native proteins apart and to translocate the denatured polypeptide into ClpP for degradation. Here, we probe polypeptide features important for translocation. ClpXP degrades diverse synthetic peptide substrates despite major differences in side-chain chirality, size, and polarity. Moreover, translocation occurs without a peptide -NH and with 10 methylenes between successive peptide bonds. Pulling on homopolymeric tracts of glycine, proline, and lysine also allows efficient ClpXP degradation of a stably folded protein. Thus, minimal chemical features of a polypeptide chain are sufficient for translocation and protein unfolding by the ClpX machine. These results suggest that the translocation pore of ClpX is highly elastic, allowing interactions with a wide range of chemical groups, a feature likely to be shared by many AAA+ unfoldases.
