Figure 1.
CDC domain organisation and mechanism of pore formation.
A. Crystal structure of the archetypal CDC PFO and its schematic representation. Domain 1 is coloured blue, Domain 2 coloured green, Domain 3 coloured red, orange and pink and Domain 4 coloured yellow. Together Domains 1 and 3 form the ‘head’ domain distantly related to the MACPF domain. Specific transmembrane regions include the TransMembrane Helices (TMH) 1 coloured orange and TMH2 coloured pink; the strand β5 and the undecapeptide loop are indicated. B. Current model of CDC pore formation. After the membrane binding event, monomers oligomerize into a ring-like structure (30 to 50 monomers; prepore). Upon formation of the oligomeric pore, both helical clusters insert into the transmembrane bilayer (grey bars) as two β-hairpins (orange and pink) part of a giant β-sheet barrel. Concomitantly Domain 1 is subject to a vertical collapse associated with a proposed “buckling” of Domain 2 (reviewed in [1], [58]).
Figure 2.
Identification of rigid fragments in CDC structures.
A & B. Structural alignment of all CDC structures and corresponding Lesk-Hubbard plot. 3.7 Å Cα-RMSD for 429 conserved positions; the common core at 1.3 Å sieving RMSD (indicated by the arrow) in B in red (228 positions). In Domain 3, the β5 strand and the TMH2 helical bundle are regions of plastic deformation. C & D. Structural alignment of all CDC Domain 2 and 4 structures and corresponding Lesk-Hubbard plot. 1.9 Å Cα-RMSD for 157 residues; common core at 0.8 Å sieving RMSD (indicated by the arrow) in red (78 positions). The undecapeptide and the protruding β-hairpin are regions of plasticity.
Table 1.
CDC crystal structures used in this study.
Figure 3.
Twist of Domain 2 and its influence on Domain 4 orientation.
A. Variations in Domain 4 orientation across the CDC family. Superposition is the same as Figure 2A. Only four representative structures are shown, which cover the entire range of Domain 4 orientation in CDCs. B. Coupling between Domain 2 twist and Domain 4 orientation. Domain 3 is omitted for clarity.
Figure 4.
PFO Domain 4 and twist of Domain 2.
A. Variations in Domain 4 orientation for the PFO structures. Superposition is the same as in Figure 2A. Only five structures are shown for clarity: PFO I (blue); PFO IIB (dark blue); PFO IIID (cyan); PFO IIIA (orange); PFO IIIC (light orange). B. Coupling between PFO Domain 2 twist and Domain 4 orientation. Domain 3 is omitted for clarity. C. Twist values as a function of amino acid pairs for the continuous strands forming Domain 2. The seven crystal conformations are classified in two groups: conformations with partial loss of Domain 2/4 interface (interface ∼425 Å2; PFO IIIA & IIIC) in orange and all the others in blue (interface ∼605 Å2; Table 2). Minimum and maximum values are displayed for each group. D. Representative mainchains of Domain 2 β-sheet in stick representation. Blue: PFO I; orange: PFO IIIA. Superposition is the same as in Figure 2A. Numbers indicate amino-acid positions, dashed lines indicate mainchain hydrogen bonds.
Table 2.
Domain 2-Domain 3 interface features and Molecular simulations performed.
Figure 5.
Plasticity and deformation of Domain 2 from PFO MD simulations.
Analysis of the different MD simulations for PFO I, PFO IIIA, PFO IIIB, PFO IIIC (indicated at the left of each panel). Left panel: distance representative of the Domain 2/TMH2 interface between residues 287 and 388 (see also Table 2). Centre and right panels: values of twist at positions discussed in the text and illustrated in Figure 4C plotted versus the distance between residues in left panel. The coloured vertical bars correspond to the range of twist values derived from the analysis of the PFO crystal structures (Figure 4B). The pairs of residues considered are indicated at the top of the figure.
Figure 6.
Flexibility of Domain 4 with respect to Domains 1–3 in MD simulations.
A. Each panel corresponds to an MD simulation. The starting conformer (cartoon representation, grey) is indicated at the left of the molecule. Cα positions taken from snapshots of the simulations are represented as dots after alignment on Domains 1–3. B. Arrows (red) indicate the directions of motion of the third slowest mode (scaled to 3.5 Å for clarity), which best describes the increased distance between Domain 2 and TMH2. The starting conformer is indicated at the left of its cartoon representation. The reasonably small fractional variance explained by the modes (11% (PFO I) and 8% (PFO IIIB), collectivity of 0.34 and 0.45 respectively) identifies a more localized nature of the motion. This suggests that such movement participates to, or enhances, the observed flexibility of Domain 4.
Figure 7.
Prepore conformation of the CDC molecule.
A. View of the CDC monomer in the prepore conformation within the cryo-EM map (transparent surface). B. Overall oligomeric arrangement within the cryo-EM map. Only the tetramer used in the modeling is shown (see Methods). C. Structural alignment of the ILY IB conformation (pale green) to the prepore conformation (pale orange) (4.4 Å; 448 positions). D. Prepore conformation in the context of representative CDC crystallographic structures.
Table 3.
Stereochemistry indicators for the atomic models.
Figure 8.
Pore conformation of the CDC molecule.
A. Cut view of the CDC monomer in the pore conformation within the cryo-EM map (transparent surface). B. Subunits arrangement in the pore (cut view). The tetramer shown is the symmetrically modeled tetramer (see Methods). Subunits have alternate colouring with Domain 2 coloured green. C. Domains 2 arrangement in the pore viewed from outside of the ring. A Domain 2 is highlighted in green sandwiched by two adjacent monomers. D. Prepore and pore conformations aligned on Domain 4. Prepore Domain 2 is in orange; pore Domain 2 is in green.
Figure 9.
Orchestrated domain movement and proposed mechanism of pore formation.
Schematic representation of the proposed new model of prepore (top row) to pore (bottom row) transition shown from three point of views: A) from outside the ring formed by the oligomeric complex, B) Top view and C) from inside the ring. The dashed line symbolizes the position of Domain 4 in the prepore complex. The arrows symbolize the concerted movement experienced by the globular head domain (Domains 1 & 3) upon membrane insertion. Alternate monomers are represented with alternate colors; only three monomers are displayed for clarity; remaining monomers are represented as a transparent surface. D. Schematic representation of proposed CDC pore formation. The monomer displays flexibility in the orientation of Domains 1–3 versus the membrane binding Domain 4. Upon self-oligomerization into the prepore complex the monomer is trapped in a monomer accessible conformation. Upon membrane insertion, the orientation of Domain 2 flattens with respect to the membrane surface. This is accompanied by a vertical collapse of Domain 1 and 3, which brings them closer to the bilayer surface and allows insertion of TMH1/2 as β-hairpins. Colours are identical to Figure 1.