Binding of the ClpA unfoldase opens the axial gate of ClpP peptidase

Abstract

ClpP is a serine protease whose active sites are sequestered in a cavity enclosed between two heptameric rings of subunits. The ability of ClpP to process folded protein substrates depends on its being partnered by an AAA+ ATPase/unfoldase, ClpA or ClpX. In active complexes, substrates are unfolded and fed along an axial channel to the degradation chamber inside ClpP. We have used cryoelectron microscopy at approximately 11-A resolution to investigate the three-dimensional structure of ClpP complexed with either one or two end-mounted ClpA hexamers. In the absence of ClpA, the apical region of ClpP is sealed; however, it opens on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops (residues 1-17) of ClpP, which tend to be poorly visible in crystal structures, indicative of conformational variability. Nevertheless, we were able to model the closed-to-open transition that accompanies ClpA binding in terms of movements of these loops; in particular, "up" conformations of the loops correlate with the open state. The main part of ClpP, the barrel formed by 14 copies of residues 18-193, is essentially unchanged by the interaction with ClpA. Using difference mapping, we localized the binding site for ClpA to a peripheral pocket between adjacent ClpP subunits. Based on these observations, we propose that access to the ClpP degradation chamber is controlled allosterically by hinged movements of its N-terminal loops, which the symmetry-mismatched binding of ClpA suffices to induce.

FIGURE 1.

Cryo-EM reconstructions of ClpP attached to 1 or 2 ClpA. a, cryo-EM showing a field of ClpAP complexes. Two side views, of a 1:1 (A P) and a 2:1 complex (A P A), are indexed. b and c, surface renderings of the ClpP-containing portion of the reconstructed 2:1 complex in side view (b) and axial view (c). d–f, surface renderings of the reconstructed 1:1 complex in side view (d) and both axial views (e and f). The boundaries between ClpA and ClpP are marked in b and d. The reconstructions were cross-cut at the levels marked to give the axial views shown in c and e.

FIGURE 2.

Fitting of an atomic model of the ClpP tetradecamer barrel (residues 18–193; PDB code 1tyf) into the cryo-EM reconstruction of the 1:1 complex. In all four panels, the cryo-EM map is shown as a transparent surface with the subunits represented as blue ribbon diagrams except for one subunit in each ring, colored in green. a, side view of the outer surface. b, cutaway side view contrasting the axial opening at the ClpA-bound end (red arrow) with the blocked axial channel at the ClpA-free end (orange oval). c and d show axial views from the ClpA-bound and the ClpA-free ends, respectively. The first residue on the head domain (residue 18) and its C terminus (residue 193) are marked on the green subunit in c.

FIGURE 3.

Different distributions of N-loops in the ClpA-bound and ClpA-free states of ClpP. a, gray scale longitudinal central sections of the 1:1 (left) and 2:1 (right) reconstructions. A marks ends where ClpA is bound. Beside the 1:1 section are schematics delineating density zones in the designated portions of the section. In the top panel (part of the ClpA-bound end of ClpP), the sleeve of N-loop-associated density is in red contour; the contacting portion of ClpA is in green, and the ClpP barrel is in black. The blue bar marks the channel-blocking density at the ClpA-free (bottom) end of ClpP. Red arrows in both sections point to the sleeve. b, gray scale longitudinal central sections simulated from crystal structures of ClpP, band-limited to 12-Å resolution and 7-fold symmetrized. Left, structure for PDB 1tyf for residues 18–193, i.e. without any N-loops. Note the wide axial channel. Right, structure for PDB 1yg6 with (left) all seven N-loops, six up and 1 down, and (right) with only the six up N-loops (Nl = N-loop). d, 1yg6 structure, as docked into the cryo-EM density map of the 1:1 complex. The six up N-loops at the ClpA-bound end are in red, and the one down N-loop is in magenta (magenta arrowhead). The partially visualized N-loops at the ClpA-free end are in magenta. c, gray scale longitudinal central sections through two models for the 1:1 complex. The top half of both models is from PDB 1yg6 with six up N-loops. The bottom halves were modified from 1yg6 in two different ways by allowing rigid body rotation of the N-loops around residues 17 and 18. They give examples of how N-loops may be packed so as to close the channel (blue bars).

FIGURE 4.

Point of contact of ClpA on the surface of ClpP. a and b, axial (a) and side view (b) representations of the interaction between the ClpA and ClpP rings. The cryo-EM density for the 2:1 complex is shown in transparent gray, and the fitted ClpP model is in ribbon diagrams (red for residues 1–17 and blue for residues 18–193). Two neighboring subunits are colored yellow and cyan. The difference map between the cryo-EM density and a 12-Å resolution rendering of the docked ClpP model (residues 18–193) is overlaid in green. c, magnification of the marked region in b, with the cryo-EM density (other than the difference density) removed for clarity. Residues forming the pore at the interface of the yellow and cyan monomers and which have side chains pointing toward the ClpA density (green) are numbered and represented as balls and sticks.