Stability, orientation and position preference of the stem region (residues 689-703) in Hepatitis C Virus (HCV) envelope glycoprotein E2: a molecular dynamics study

Input structure preparation

The protein sequence of HCV E2 genotype 1a (H77 strain) (Uniprot ID P27958) used to prepare the models for MD simulations was obtained from UniProtKB/Swiss-Prot database (www.uniprot.org)¹⁰. The following is the sequence segment of HCV E2 used to prepare the first model for this work, referred to as an ideal helix model: ⁶⁸³PALSTGL ⁶⁹⁰IHLHQNIVDV ⁷⁰⁰QYLYGVGSSI ⁷¹⁰ASWAIKWEYV ⁷²⁰VLLFLLLADA ⁷³⁰RVCSCLWMML ⁷⁴⁰LISQAEA. The ideal helix model was generated using Pymol (http://pymol.sourceforge.net) by orienting the ST segment (residue 683–714) perpendicular to the TM domain (residues 715–746). The protein structure images in this work were prepared by the Pymol program. The I-Tasser webserver¹¹ was used to generate the second model. The complete sequence of E2 was submitted to the I-Tasser server. Five models were generated and the model with a low root mean square deviation (RMSD) with the available NMR structure (2KZQ.pdb) and consisting of a helical TM domain was selected. Only the same segment as examined with the ideal-helix model was used for further MD simulations. The first model of the E2 NMR ensemble (PDBID: 2KZQ) retrieved from the Protein Databank (PDB)¹² was used in the third simulation system. This E2 protein segment was based on the HCV genotype 2a (JFH-1) (Uniprot ID Q99IB8)⁹.

System preparation

Pre-equilibrated dipalmitoylphosphatidylcholine (DPPC) lipid bilayer was retrieved from the web of Prof. Tieleman (http://moose.bio.ucalgary.ca/). Peptide orientation in DPPC lipid bilayer was done by aligning the hydrophobic belt of the peptide, parallel to the membrane plane using LAMBADA¹³. The optimal number of overlapping lipid molecules was subsequently calculated and removed followed by lipid expansion (inflation) and alternating twenty steps of deflation and energy minimization to allow the peptide to be embedded within the bilayer using inflateGRO2¹³. A short 100 ps energy minimization was employed to relax possible steric conflicts. Ions and counter ions were added to neutralize the system followed by 20 ns position-restrained simulation allowing the bilayer to re-equilibrate around the protein. Production MD simulations were carried out for 100 ns in the I-Tasser model, and 20 ns for both the ideal helix model and NMR structure (2KZQ.pdb).

MD simulations

The DPPC lipid bilayer interactions were described using the Berger force-field parameters¹⁴. The TM helices were modeled with the united atom force-field GROMOS96 53a6¹⁵. Simulations were performed with the Gromacs 4.5.5 package¹⁶ using 2-fs time steps. Periodic boundary conditions were used in all directions. Bonds to hydrogen atoms were constrained using the LINCS algorithms¹⁷. For the short-range van der Waals interactions, a cut-off distance of 1.0 nm was used. The long-range electrostatic interactions were treated using the particle mesh Ewald (PME) method with a grid spacing of 0.12 nm and cubic interpolation. The non-bonded pair list was generated every 10 steps with a cut-off of 1.0 nm. Water, lipid and peptide systems were coupled separately to temperature baths with 323 K for the DPPC using the Berendsen algorithm with a time constant of τ_T = 0.1 ps¹⁸. To maintain constant pressure, semi-isotropic coupling was employed separately for the lateral and for the normal directions with Berendsen weak coupling and a τ_p = 1 ps time constant. The compressibility was set to 4.5 × 10^-5 bar^-118.

Analyses of the trajectories were primarily performed with tools included in the Gromacs 4.5.5 suite¹⁶. RMSD analyses were based on the coordinates of all atoms of the peptides. The bilayer thickness was measured by averaging the distances between lipid headgroups in the upper and lower leaflets of the lipid membrane with the tool GridMAT-MD⁹.

Stability of the amphipathic segment

A stable helical conformation of the E2 ST region (residue 689–703) was consistently observed with some uncharacteristic spikes in the early stages and towards the end of the MD simulation of the I-Tasser model and the NMR derived structure (2KZQ.pdb), respectively. RMSD of this amphipathic segment was also consistently observed to be progressing within the commonly accepted 2 Å range for the ideal helix and NMR structures throughout the simulations. On the other hand, the I-Tasser model showed subtly higher RMSD progression over the simulation time (Figure 1, Figure 2 and Figure 3a, 3b).

Figure 1. Superimposition of the amphipathic region of E2 (segment 689 to 703) in three molecular dynamics simulations.

The most stable helical region of the E2 amphipathic segment during molecular dynamics simulations. 2KZQ in red, I-Tasser model in black and ideal helix model in gray.

Figure 2. Helix length and RMSD of I-Tasser model.

Both amphipathic regions and transmembrane domain located in the hydrophobic core of the bilayer. Length of helix (residues 689–703) is plotted in red, root mean square deviation of the same residues with respect to the starting structure is plotted in black.

Figure 3. Helix length and RMSD of the ideal helix model and 2KZQ structures.

(a) Molecular dynamics simulation of the ideal helix model. The amphipathic segment was positioned in water-lipid interface and the transmembrane domain was oriented in the hydrophobic core of the bilayer. The length of the helix (segment residue 689–703) is plotted in red, root mean square deviation (RMSD) of the same residues with respect to the starting structure is plotted in black. (b) Molecular dynamic simulation of the nuclear magnetic resonance derived structure (2KZQ.pdb). 2KZQ was positioned in the water-lipid interface. The length of the helix segment is plotted in red; RMSD of the same residue with respect to starting structure is plotted in black.

Secondary structure stability observed in these three contrasting simulation systems can be attributed to the amphipathic nature of the residues allowing the helix to retain its structure on both the hydrophobic core of the lipid bilayer and the hydrophilic environment of the solvent. Furthermore, secondary structure analyses done by Albecka et al. predicted that the amino acid segment 689–703 consists of more than 50% helix. In addition to that, consistent results were shown from CD spectra and NMR with 50% 2,2,2-trifluoroethanol (TFE) environment, which both detected the presence of helical segments in the E2-ST region. The most amphipathic helix characteristics are displayed in the E2-ST segment 687–703. Based on the profile, Albecka et al. speculated that this segment is a helix upon membrane binding⁹. Interestingly, the presence of the TM domain in our simulation does not appear to contribute significantly to the helix stability of the amphipathic region.

The helical length of this region does not vary much between the I-Tasser and the ideal helix models (Figure 2 and Figure 3a), which include both the ST region and TM domain, compared with the 2KZQ.pdb (Figure 3b) which does not include the TM domain. This observation suggests that lipid-peptide interactions play a larger role in stabilizing the secondary structure of this amphipathic segment compared with the TM domain. These data provide evidence of the contribution of lipid to structural stability modulation and are in good agreement with the hypothesized lipid and/or protein contribution to structural stability⁹. The higher RMSD value observed in the I-Tasser model simulation was well anticipated and was mainly attributed to the relative positioning of the ST region, which was sandwiched in between lipid leaflets, forcing the segment to reorganize its structure conformation and having only a minimal effect on the helical integrity of the secondary structure. Examining this reorganization further by monitoring the distance of the amphipathic segment to lipid leaflets led to another interesting observation described in the next section of this article.

Orientation and positional preference of the amphipathic segment

Monitoring the movement of the amphipathic segment during the simulations led to another interesting observation. Segment of residues 689–703 in the I-Tasser model appeared to move towards the hydrophobic core of the lipid bilayer as depicted by steady progression in the distance to both the upper and lower lipid leaflets depicted in Figure 4. This reorganization is surprising and interesting because we would have previously assumed that the amphipathic region exposed to the solvent would hold the structure steady, despite some part of the amphipathic region being initially vertically positioned in the hydrophobic core of the lipid (Supplementary Figure 1). In addition, given the amphipathic nature of the residues in this segment, one could postulate that the residues would remain in this position. However, over the period of the simulation the segment reoriented by moving away from the lipid leaflets, while at the same time retaining structural integrity. The structural stability of the segment, as discussed in the previous section of this article, is attributed to the amphipathic nature of the residues but this does not explain the movement towards the hydrophobic core of the lipid. The systematically orchestrated movement towards the hydrophobic core of the lipid leaflets indicates a strong orientation preference of the amphipathic segment, which in this specific case was parallel to the lipid leaflets. We then monitored the segment movement relative to the lipid leaflets with the other two simulations (the ideal helix model and the 2KZQ structure). The segment was initially positioned horizontally to the lipid leaflets. The results showed that the distance of the amphipathic segment in both simulations was consistently within 4 Å to the lipid phosphate head group throughout the simulation (Figure 5). These data further clarify the orientation preference of the amphipathic segment with respect to the lipid leaflets and suggest that the residues are positioned at the lipid-water interface in a very stable manner. Interestingly, Albecka et al.⁹ speculated that these residues could have an in-plane topology or orientation and suggested that the ST region would ideally be positioned at the membrane lipid-water interface, which is again in agreement with our data⁹.

Figure 4. Distance between the helix (residues 689–703) and lipid leaflets during molecular dynamic simulation of the I-Tasser model.

The distance to the upper lipid leaflet is plotted in red and lower leaflet is plotted in black.

Figure 5. Distance of the amphipathic segment to the lipid leaflets.

The 2KZQ is colored in black while the ideal helix model is colored in red. Amphipathic residues remain within 4 Å from the phosphate head group throughout the simulations.

Residue contributions and relevance

Having observed a clear preference of amphipathic segment orientation in the lipid bilayer environment, it was also pointed out to us that it would be desirable and interesting to look into residue contribution on this behavior. We then plotted the residue distribution in the lipid water interface for the 2KZQ structure and ideal helix model (Supplementary Figure 2) to identify specific residues positional preference in the lipid water interface. As depicted in Supplementary Figure 2, a clear residue preference in lipid-water interface was not observed. We found polar and charged residues distributed in both hydrophilic and hydrophobic regions of the interface; a similar observation was also found for non-polar residues. This result indicates the overall contribution of the residues played more roles in the stability and positional preference of this segment in lipid-water interface superseding any individual residue contribution. Characterizing the residue distribution in our I-Tasser model would not provide information on the residues preferences since the segment is situated exclusively in a hydrophobic environment. To get some insight on this segment, residue rotation analysis of this segment coupled with qualitative visualization of polar, nonpolar and charged residues was carried out (Supplementary Figure 3). Synchronized rotation of the amphipathic segment was observed and convergence was captured after 50 ns and for the remaining period of the simulation. In contrast to the ideal helix model and 2KZQ structure simulations, a clear preference to hydrophilic and hydrophobic regions was observed. Polar and charged residues were oriented towards the hydrophilic region and non-polar residues were oriented towards the hydrophobic region. We also noticed that convergence in rotation on 50 ns (Supplementary Figure 3) coincide nicely with a major shift in distance profile (Figure 4), which also started at around 50 ns and stabilized at around 70 ns. This indicates that the reorganization of this segment correlates tightly to the residues’ preference towards hydrophilic or hydrophobic environments. It is interesting to highlight that these residues’ preference to hydrophilic and hydrophobic regions in lipid-water interface (ideal helix model and 2KZQ structure) appears to be loosely correlated (no clear preference observed) in contrast to the residues’ preference in environments exclusively on lipid (I-Tasser model) where a clear preference was observed. Due to the fact that the movement of the I-Tasser model residues is synchronized, we found no evidence to substantiate major contribution of individual residues to this structural rearrangement. Overall hydrophilic and hydrophobic residue contribution to the segment reorientation and reorganization is further highlighted.

Limitations and future work

An orientational preference parallel to the lipid leaflets is a common feature of amphipathic helix segments. Parallel orientation of amphipathic segments has been characterized in experimental works²⁰. Amphipathic segments have also been observed to only minimally penetrate and contribute to lipid bilayer perturbation^21,22. In addition to that, lipid shape has been demonstrated to contribute to amphipathic segment topology in the lipid interface²³. Taking all this information collectively, the amphipathic segment of residues 689–703 is anticipated to be positioned stably in the lipid-water interface in a parallel topology with respect to the lipid leaflets. In line with this anticipation, the 20 ns simulations of two of our structures (ideal helix model and 2KZQ) demonstrate a consistent helical form oriented parallel to the lipid leaflet throughout the simulation period. Interestingly, our third simulated structure (I-Tasser model) demonstrated a clear preference to parallel orientation despite the fact that its initial amphipathic segment configuration was perpendicular to the lipid leaflet. We decided to subject the I-Tasser model to longer simulation (100 ns) compared to the other two structures (20 ns) mainly due to the position of its amphipathic segment. The I-Tasser model positioned the amphipathic segment sandwiched in the bilayer leaflets, in contrast to the commonly assumed position in the membrane interface. On the other hand in the ideal helix and 2KZQ models it is readily positioned at the membrane interface. True to our expectation, meaningful observations such as amphipathic segment reorganization only started to be captured after 70 ns. This highlights the importance of optimum conformational space exploration in the simulation. Despite the consistent stability observed throughout our ideal helix model and 2KZQ structures, a longer simulation time is desirable to further reaffirm the observation. Simulation of the amphipathic segment in the water-only environment is also desirable to elucidate the segment behavior. These will be added as part of the future agenda of this work.