Cryo-EM structure of the ClpXP protein degradation machinery

Abstract

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamerhexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXP's complex mode of action and provides a structural framework for future pharmacological applications.

Conformational Plasticity of the ClpAP AAA+ Protease Couples Protein Unfolding and Proteolysis

“…A number of proteolysis machines operate as hexamer:heptamer assemblies 3,4 , thus a key question emerges as to how this mechanism is conserved. Indeed, recent structures of ClpXP reveal its IGF loops are similarly flexible, supporting a conserved rotary mechanism 56 . Notably, assembly of the eukaryotic Rpt and archaeal PAN AAA+ hexamer with its 360 respective 20S core involves engagement by C-terminal HbYX motifs and gate-opening of the 20S 57,58 .…”

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

“…To date, the structure of the ZBD has not been crystallized with the rest of the protein due to its apparent disorder; yet, the ZBD of ClpX is important for its function in other bacteria to, for example, recognize specific substrates (24, 56, 57). We hypothesize that the TSSTSSP residues may serve a function in flexibility (58, 59) or extension of the N-terminus, which in turn may modulate its unfoldase/chaperone activity (60). Interestingly, an SP motif has been implicated in initiation of a Type I β-hairpin turn (61, 62), which may serve as a mechanism through which the ClpX Ctr N-terminus adopts a unique conformation to recognize uncharacterized adaptors.…”

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

“…Crystal structures show that ADEP binding to ClpP results both in opening of the axial pore and assembly of its N-terminal sequences into a collar of β-hairpins (Li et al, 2010;Lee et al, 2010b). Based on a recent cryo-EM structure of Listeria monocytogenes ClpXP, it was concluded that ClpX binding does not induce the same widening of the ClpP pore as does ADEP binding (Gatsogiannis et al, 2019). By contrast, our results support the opposite conclusion, as the diameter and overall structure of the ClpP pore in our E. coli ClpXP structures were extremely similar to the crystal structure of ADEP-activated E. coli ClpP (Li et al, 2010), with an overall RMSDs of 0.8 Å for all Cα's in a single ClpP ring and 0.6 Å for all Cα's of the seven N-terminal β-hairpins and adjacent α-helices that define the pore diameter.…”