Fig 1.
Septin structure and initial configurations of the extended AH with a lipid bilayer.
(A) PDB structure of a nanoscale septin oligomer with a zoomed-in view of the Cdc12 subunit and its Amphipathic Helix (AH) domain with adjacent residues. The extended AH regions are color-coded: N-terminus (blue), AH domain (green), hydrophobic residues (magenta), and C-terminus (red). The corresponding amino acid sequence is also shown. (B) Initial configuration of the unbound state: the extended AH peptide is placed in solution above the lipid bilayer. (C) Initial configuration of the bound state: the extended AH is shallowly inserted into the membrane. The AH hydrophobic face is aligned with the lipid tails. Lipid hydrophobic tails are shown in cyan and phosphate headgroups in dark blue. Lipids are rendered semi-transparent to highlight the peptide orientation and residue types.
Fig 2.
Bound peptide adopts a stable and smile-like curvature in the membrane.
(A) MD simulation snapshot of a membrane-bound extended AH, with the N-terminus in blue and the C-terminus in red. Arrows indicate hydrophobic residues in the extended regions that face outward toward the solution, unlike the hydrophobic face of the AH region that orients inward. (B) Snapshot of the spatial map of area per lipid (APL) in the membrane upper leaflet. The presence of the peptide increases local APL near its binding site. The dots indicate the position of the backbone alpha carbons. (C) Peptide conformation color-coded by local bending angle using the Bendix tool. (D) Residue-resolved local bending angle (blue, left axis) and corresponding radius of curvature (red dashed, right axis) corresponding to the Bendix snapshot. (E) The local helix bending angle map over simulation time shows stability in the local bending of the membrane-bound peptide. N-terminal residues bend more than those on the C-terminal side.
Fig 3.
The presence of a membrane-bound peptide modulates the positioning and orientation of the unbound peptide toward the membrane.
(A) Distribution of the Z-component of the center of mass (COM) for the unbound peptide in systems with single (orange, n = 4) and two peptides (green, n = 4). The gray curve shows the distribution of Z-positions of lipid phosphate headgroups. Left inset: MD simulation snapshot showing the single unbound peptide approaches the membrane via the C-terminus (orange). Right inset: MD simulation snapshot showing an unbound and a bound peptide. The N-terminal region of the unbound peptide can interact with the bound peptide (green). (B) Box plot of the Z-COM of residues in the system with a single unbound peptide (n = 4). The blue and red dashed lines separate the N- and C-terminal regions, respectively. (C) Box plot of the Z-COM of residues of the unbound peptide in the multiple-peptide system. The first four N-terminal residues (blue box) show a closer approach to the membrane, consistent with interaction with the membrane-bound peptide.
Fig 4.
Salt bridge formation between charged amino acids promotes peptide-peptide interactions.
(A) Contact map showing intra- and inter-peptide contacts. The zoomed-in inter-peptide region highlights a salt bridge between charged residues R4 and E21. The N-terminal of the floating peptide preferentially interacts with the ending region of the AH domain of the bound peptide, indicating an antiparallel peptide orientation. (B) The domain-based coarse-grained contact maps averaged over all replicas in different time windows show two peptides interacting in an anti-parallel configuration that reaches a steady state. (C) Simulation snapshot showing multiple salt bridges formed between charged residues of the floating and bound peptide. Basic residues are shown in blue, acidic in red, polar in green, and hydrophobic in magenta. (D) number of inter-peptide contacts between residue pairs of the floating and bound peptides. The majority of contacts involve charged residues. (E) Time evolution of residue pair distances for the top-ranked salt bridge contacts, revealing how the two peptides interact (replica 2). R and E residues form a stable salt bridge throughout the simulation.
Fig 5.
The position of bound peptides and the lipids arrangement change in single- and multiple-peptide systems.
(A and B) Top and side view simulation snapshots of systems with the same initial structure of two parallel bound peptides show different behavior: (A) Both peptides remain bound in 400 ns simulation time (light green, 75% of replicas) (B) One of the peptides dissociates from the bilayer and interacts with the other peptide. (dark green, 25% of replicas). (C) Z-component of the center of mass distribution of the bound peptide in different systems. Inset: Simulation of snapshots of a single membrane-bound peptide (orange) and a system of bound and unbound peptides with lipid bilayer (red). (D) Spatial map of area per lipid (APL) in the membrane upper leaflet for two bound peptides initially placed in parallel (the non-favorable) orientation. The two peptides increase the local APL near the binding site and compact the distant lipids. The dots indicate the position of the backbone alpha carbons with N-terminal residues in blue and C-terminal residues in red. (E) Histogram of the lipids order parameter for systems with a single (orange) and two (green) bound peptides in the vicinity of (solid line) and far from (dashed line) the binding site.
Fig 6.
Proposed mechanism of septin polymerization on the curved lipid bilayer.
(A) The bound septin facilitates the binding of neighboring septins through salt bridge interactions between their AH domains in anti-parallel, forming septin assemblies. (B) Multiple sequence alignment and sequence logo of the Cdc12 amphipathic helix domain across fungal species. Conserved charged residues implicated in inter-peptide salt bridges are highlighted, including arginine residues in the N-terminal ERIR motif and glutamic acid residues in the LEE motif. These charged residues are instrumental to salt bridge interactions and peptide-peptide contacts.
Table 1.
Summary of molecular dynamics simulations.