Computational studies of colicin insertion into membranes: the closed state

Abstract

Colicins are water-soluble toxins that, upon interaction with membranes, undergo a conformational change, insert, and form pores in them. Pore formation activity is localized in a bundle of 10 α-helices named the pore-forming domain (PFD). There is evidence that colicins attach to the membrane via a hydrophobic hairpin embedded in the core of the PFD. Two main models have been suggested for the membrane-bound state: penknife and umbrella, differing in regard to the orientation of the hydrophobic hairpin with respect to the membrane. The arrangement of the amphipathic helices has been described as either a compact three-dimensional structure or a two-dimensional array of loosely interacting helices on the membrane surface. Using molecular dynamics simulations with an implicit membrane model, we studied the structure and stability of the conformations proposed earlier for four colicins. We find that colicins are initially driven towards the membrane by electrostatic interactions between basic residues and the negatively charged membrane surface. They do not have a unique binding orientation, but in the predominant orientations the central hydrophobic hairpin is parallel to the membrane. In the inserted state, the estimated free energy tends to be lower for the compact arrangements of the amphipathic helix, but the more expanded ones are in better agreement with experimental distance distributions. The difference in energy between penknife and umbrella conformations is small enough for equilibrium to exist between them. Elongation of the hydrophobic hairpin helices and membrane thinning were found unable to produce stabilization of the transmembrane configuration of the hydrophobic hairpin.

Figure 1

Three-dimensional structures of the PFD of the colicins studied in this work (cartoon representations were made using VMD84). The groups to which they belong are also indicated. The hydrophobic hairpin in each case is highlighted. The arrows show the location and direction of the pulling force applied to get an umbrella model (see Methods Section).

Figure 2

Side and upper views the a) Compact, b) Loose-Array and c) Circular model for the umbrella conformation. (Cartoon representations were made using VMD84). The horizontal lines represent the membrane surface. These structures correspond to the initial structures of the simulations run for the inserted state of colicins.

Figure 3

Distribution of the values of the angle formed by the hydrophobic hairpin and the membrane. In the upper graph the results at low pH are shown and in the lower part, those for pH=7. The direction of the axis of the hydrophobic hairpin (highlighted in black) is shown as an inset of the lower graph as well as the angle corresponding to the abscissa axis (the membrane surface is represented by a horizontal line). An example of the adsorbed state with the hydrophobic hairpin oriented parallel to the membrane plane is shown as an inset in the upper graph (cartoon representations were made using VMD84).

Figure 4

Qualitative comparison of the inter-helical distance distributions of colicin A in our simulations with the EPR spectroscopy results reported by Böhme et. al.. The green lines correspond to the experimental results, while the black, red and blue ones correspond to the simulation results obtained for the compact, loose-array and circular models, respectively.

Figure 5

Representative snapshots for the penknife and umbrella conformations in the inserted state of the four colicins studied here. The arrows point to the loop that becomes more extended in the umbrella model of colicin Ia. The line representation highlights the aromatic and acidic residues representative in the interaction with the membrane of the umbrella conformations of colicins Ia and E1 (see discussion about the helix elongation process).

Figure 6

Representative snapshots of the penknife and umbrella conformations in the inserted state of colicins Ia and E1 with a longer hydrophobic hairpin. The line representation highlights the aromatic and acidic residues representative in the interaction with the membrane of the umbrella conformations of colicins Ia and E1 (see text).

Figure 7

Variation (delta) with respect to the system with T=26 Å of the effective energy (discontinuous line) and the effective plus the deformation free energy (continuous line) with membrane thickness.

Figure 8

Mechanism of colicin insertion proposed by Kienker and coworkers and supported by the results presented in the present paper. Adapted from Fig. 7 in reference . “ADS” stands for the adsorbed state; ΔG_ADS-P is the difference in free energy between the penknife conformation and the adsorbed state, and ΔG_PU is the difference in free energy between the umbrella and the penknife conformations.