Insertion and assembly of membrane proteins via simulation

Abstract

Interactions of lipids are central to the folding and stability of membrane proteins. Coarse-grained molecular dynamics simulations have been used to reveal the mechanisms of self-assembly of protein/membrane and protein/detergent complexes for representatives of two classes of membrane protein, namely, glycophorin (a simple alpha-helical bundle) and OmpA (a beta-barrel). The accuracy of the coarse-grained simulations is established via comparison with the equivalent atomistic simulations of self-assembly of protein/detergent micelles. The simulation of OmpA/bilayer self-assembly reveals how a folded outer membrane protein can be inserted in a bilayer. The glycophorin/bilayer simulation supports the two-state model of membrane folding, in which transmembrane helix insertion precedes dimer self-assembly within a bilayer. The simulations also suggest that a dynamic equilibrium exists between the glycophorin helix monomer and dimer within a bilayer. The simulated glycophorin helix dimer is remarkably close in structure to that revealed by NMR. Thus, coarse-grained methods may help to define mechanisms of membrane protein (re)folding and will prove suitable for simulation of larger scale dynamic rearrangements of biological membranes.

Figure 1

Atomistic (left hand) and coarse grained (right hand) models compared for: A a DPC molecule; and B a GpA helix. Colours for atoms: cyan = carbon; red = oxygen; blue = nitrogen; bronze = phosphorus; yellow = sulphur. Colours for CG particles: cyan = apolar; red = polar; blue = positively charged; bronze = negatively charged; yellow = neutral.

Figure 2

Self-assembly of protein/detergent (DPC) micelles. The upper row shows snapshots from a simulation of OmpA/DPC; the lower row shows snapshots from a simulation of GpA/DPC. In each case DPC is in ‘bonds’ format with the hydrophobic tail in cyan and the polar head in red. The protein is shown as a blue Cα trace (OmpA) or blue and purple Cα traces for the two GpA monomers.

Figure 3

Detergent radius of gyration for OmpA (black lines) and GpA (grey lines) self-assembly into protein/detergent micelles. The thin lines correspond to all detergent molecules, whereas the thick lines correspond to the main micelle (i.e. the detergent molecules in the ‘globule’ are excluded). The dashed lines correspond to the radii of gyration of equivalent micelles from atomistic simulations . The kinetics of formation of the main micelle have been characterised by measuring the exponential decay in radius of gyration, yielding time constants of ~5 ns for GpA and ~12 ns for OmpA. These are similar to the values from corresponding atomistic simulations.

Figure 4

Root mean square fluctuation (RMSF) for Cα atoms vs. residue number for OmpA simulations. A OmpA/micelle, comparing AT = atomistic (pre-formed) (grey line) and CG = coarse-grained (black line) simulations. B OmpA/bilayer, comparing AT = atomistic (grey line) and CG = coarse-grained (black line) simulations. Note that in both cases the atomistic RMSFs have been multiplied by a factor of 3 to normalise for the increased sampling in the longer CG simulations. This was based on comparison of the mean square fluctuations vs. sample time for the uncorrected atomistic simulations and the coarse-grained simulations.

Figure 5

Self-assembly bilayer and insertion of an OmpA molecule. The lipid (equivalent to DPPC) is shown in ‘bonds’ format with the hydrophobic tails in cyan, the glycerol backbone in green, and the polar head in red. The Cα trace of the OmpA molecule is in blue. Water particles are omitted for clarity. Snapshots from a simulation of duration 200 ns are shown, at t = 0, 2, 8 and 12 ns.

Figure 6

Self-assembly of a GpA helix dimer in a lipid bilayer. The lipid is shown in ‘bonds’ format with the hydrophobic tails in cyan, the glycerol backbone in green, and the polar head in red. The two GpA monomers are shown as a blue and purple Cα traces. The helices do not appear to fully span the bilayer at 440 ns as a result of the simplified backbone representation. However, the CG potential results in a rather large particle diameter of 4.7 nm, as indicated in Fig. 1, and hence the interfacial bilayer region and the helical termini do actually overlap (see Fig. 10).

Figure 7

Time course of GpA TM helix dimer assembly. A Trajectories of the sidechain particles for Thr74 (black lines) and Ser92 (grey lines) along the bilayer normal axis. The solid lines correspond to one GpA monomer, and the broken lines to the other monomer. The equilibrium locations of the glycerol backbone of upper and lower membrane leaflets (once the bilayer has formed, i.e. after ~25 ns) are shown as horizontal black lines. Thus the centre of the bilayer is at ~4.8 nm. B Crossing angle of helices (black line; left hand axis) and the inter-helical separation distance (grey line, right hand axis) as a function of time. The inter-helical distance is calculated as that between the centres of mass of all CG particles in each helix. The corresponding crossing angles and inter-helical distances (all atoms centres of mass) in the corresponding NMR structures are 41° and ~0.9 nm , and 35° and ~0.9 nm .

Figure 8

Time course of the long (5 μs) GpA TM helix dimer self-assembly simulation. A Trajectories along the bilayer normal (z) of the sidechain particles for Thr74 of the two helices (colours correspond to those in D). The centre of the bilayer is at z ~5.5 nm. B Inter-helical separation distance. C Schematic of the helix monomer/helix dimer equilibrium, derived from the data in A,B. D Snapshots of the system, showing the two helices (using the same colour convention as in A) as Cα traces, with the bilayers represented via spheres for the lipid headgroups.

Figure 9

Structures of the GpA TM helix dimer showing (from left to right): the NMR structure in a micelle (1AF0), in a bilayer (SS), and from the CG simulation of the truncated helices (CG-tr), and from the CG simulation of the extended helices (CG-ext). In each case, the C-termini are at the top of the diagram.

Figure 10

Atom number density profiles along the bilayer normal for the A GpA/bilayer-AT and B GpA/bilayer-CG simulations. In each case the density profiles for water (W, solid black line), lipid headgroups (HG, broken black line), lipid tails (T, grey line), and protein (P, black filled region) are shown. In B the protein number density was multiplied by 3 to correct for the difference in cross-sectional area of the CG and AT simulation boxes.