Molecular dynamics studies of the archaeal translocon

Abstract

The translocon is a protein-conducting channel conserved over all domains of life that serves to translocate proteins across or into membranes. Although this channel has been well studied for many years, the recent discovery of a high-resolution crystal structure opens up new avenues of exploration. Taking advantage of this, we performed molecular dynamics simulations of the translocon in a fully solvated lipid bilayer, examining the translocation abilities of monomeric SecYEbeta by forcing two helices comprised of different amino acid sequences to cross the channel. The simulations revealed that the so-called plug of SecYEbeta swings open during translocation, closing thereafter. Likewise, it was established that the so-called pore ring region of SecYEbeta forms an elastic, yet tight, seal around the translocating oligopeptides. The closed state of the channel was found to block permeation of all ions and water molecules; in the open state, ions were blocked. Our results suggest that the SecYEbeta monomer is capable of forming an active channel.

FIGURE 1

Simulated system of SecYEβ in a lipid bilayer/water environment. SecYEβ is shown in cartoon representation with SecY, SecE, and Secβ colored in gray, orange, and ochre, respectively. The plug, transmembrane domain 2a of SecY (residues Ile⁵⁵ to Gly⁶⁵) is presented in red, and TM2b (residues Gly⁷⁶ to Ser⁹¹) and TM7 (residues Asn²⁵⁶ to Gly²⁸⁰) are shown in green (both also of SecY). The lipids are seen in yellow licorice representation with the phosphorus, nitrogen, and an oxygen of the headgroup highlighted as spheres colored in tan, blue, and red, respectively. The water box is drawn in transparent blue surface representation. (A) Side view of the simulated system. To display the protein more clearly, some lipids and water molecules have been removed, leaving a flat outward face. (B) Top view of the simulated system. The top solvation layer has been removed.

FIGURE 2

Time dependence of the RMSD of the simulated system. The RMSD relative to the crystal structure (calculated for the protein backbone) is shown for simulation sim0 (see Table 1). Three stages of the simulation corresponding to different harmonic restraints (see Methods) are clearly discernable through jumps seen at t = 0 ns, t = 0.5 ns, and t = 1.5 ns.

FIGURE 3

Translocation of deca-alanine through SecYEβ. The figure shows the results of simulation sim1 (see Table 1). The representation and coloring of SecY, SecE, and Secβ is the same as in Fig. 1 with the exception that SecE and Secβ are rendered transparent. The plug (TM2a of SecY, residues Ile⁵⁵ to Gly⁶⁵) is shown in red. (A) Front view of SecYEβ and deca-alanine at t = 0. Deca-alanine is shown in blue cartoon representation together with its (transparent) surface and is positioned on the cytoplasmic side of SecYEβ before translocation. (B) Top view of the SecYEβ-deca-alanine system at t = 0.8 ns. The pore ring (residues Ile⁷⁵, Val⁷⁹, Ile¹⁷⁰, Ile¹⁷⁴, Ile²⁶⁰, and Leu⁴⁰⁶ of SecY), expanded from its equilibrium (t = 0) state, is shown in surface representation colored yellow with deca-alanine passing through it. Deca-alanine is partially unfolded at this point. (C) Final state of translocation (t = 1.4 ns). The plug has been pushed out into the solvent, and deca-alanine is seen unfolded next to the plug.

FIGURE 4

Force profile along the SecYEβ translocation pathway. (Bottom) Force versus distance along the z axis for simulations sim1, sim1a, and sim2 shown in blue, black, and red, respectively. The shaded area represents the location of the hydrophobic core of the membrane. (Top) Side cut of SecY from a representative simulation shown in surface representation. The channel is positioned sideways with the cytoplasmic side to the left and the periplasmic side to the right. The scale of the figure corresponds to that of the channel distance shown and the dashed lines correlate specific positions between the top and bottom. Highlighted are the pore ring (yellow) as well as the plug (red). TM2b and TM7 are shown in green. The figure shows a snapshot of the channel during a translocation event. The pore is represented in blue and was calculated by the program HOLE (see Methods).

FIGURE 5

Dynamics of pore formation in SecYEβ. Shown are results of simulation sim2 (see Table 1). Helices TM2b and TM7 are highlighted in green in surface representation; the SecY plug (see Fig. 1) is shown in red cartoon representation, whereas the rest of the protein is shown in transparent surface representation in the same colors as in Fig. 1. Blue spheres indicate the local pore radii as calculated by the program HOLE (see Methods). The center of the translocating helix is shown as a black sphere. (A) Channel state at t = 0. The blue spheres indicate the channel is closed at this stage, both by the pore ring and the plug. (B) Channel state at t = 1 ns. The pore is widened by the translocating helix; however the plug still partially blocks the channel. (C) Channel state at t = 1.9 ns. The plug is no longer blocking the channel. In all cases, the pore (blue) is directly adjacent to TM2b and TM7.

FIGURE 6

Relaxation of SecYEβ after translocation shown in Fig. 4. The protein is viewed from the cytoplasmic side. Shown are the results of simulation sim3 (see Table 1). The representation of the protein and plug is the same as in Fig. 1. (A) SecYEβ, immediately after translocation of deca-alanine (sim1). The plug is still outside the channel. (B) SecYEβ, 1.4 ns after the translocation. The pore ring, shown in surface representation and colored yellow, is nearly closed. (C) SecYEβ 3.6 ns after helix translocation. The plug has retracted back into the channel, effectively blocking it again.

FIGURE 7

Deformation of SecYEβ during two translocation and relaxation cycles. Shown is the RMSD (in relation to the crystal structure) versus time for the backbone of SecYEβ in simulations sim1 and sim3 (shaded) and simulations sim2 and sim4 (solid). Sim1 ends and sim3 begins at 1.4 ns, whereas sim2 ends and sim4 begins at 1.9 ns.

FIGURE 8

Back-to-back dimerization of SecYEβ. (A) Dimer of SecYEβ viewed from the periplasm. Each monomer is shown in the same representation as in Fig. 1 with the exception of the color scheme of the left monomer (blue for SecY, yellow for SecE, and red for Secβ). The plugs in the dimer are without well-defined helical structure and are shown in green. (B) Destabilization of the plugs. The RMSD of the plugs (residues Ile⁵⁵ to Gly⁶⁵) is shown after fitting the entire SecY to the crystal structure at each point. Presented in black is the RMSD for the plug of the monomer during sim0 (see Table 1); shown in red and blue are the RMSD values for each plug from the dimer as evaluated during simulation sim5.