The MD simulations provide a consistent and energetically reasonable pathway for cholesterol exchange by Osh4. Equilibrium and steered MD simulations carried out with different force constants and steering velocities (Osh4-cho SMD, SMD2−5) lead to qualitatively similar pictures. Cholesterol dissociates from Osh4 in a stepwise fashion. The upper bounds to the free energy barriers encountered for the main steps, 6 and 8 kcal/mol in the Osh4-cho SMD simulation selected for analysis are not prohibitive. Indeed, cholesterol transfer rates of ~0.15 s−1
) were recently obtained experimentally, roughly consistent with a combined barrier of ~14 kcal/mol for a pre-factor of about 1 ns−1
During the forced cholesterol unbinding, there is little change to the overall structure of Osh4, excluding those regions shown experimentally to be conformationally labile. First, water-mediated hydrogen bonds at the bottom of the pocket are disrupted. Second, the lid opens to allow access to solvent. Third, cholesterol slides out of the tunnel, making and breaking transient contacts with the tunnel wall, tunnel rim, and the hydrophobic face of the lid as it does so.
The helix α7 is the region that moves the most between the bound and apo crystal structures of Osh4 11
. One of the objectives of this study was to investigate whether the movement of α7 is coupled to lid opening. The lid and helix α7 lie next to each other, which suggested to us that α7 might in some way regulate, or be regulated by, the conformation of the lid. The conformation of α7 observed in the apo structure appeared to us to be more compatible with membrane binding, because it fits the profile of a flat membrane-docking surface surrounding the tunnel opening. In the equilibrium MD simulations of both the complex (Osh4-cho NPT) and the ligand-free form (Osh4 NPT), helix α7 is highly mobile. The C-terminus of the helix pivots around its N-terminus towards and away from the lid through out the simulation for combined distance of about 15 Å. It is the only major region of secondary structure in the complex form that is not stable in the starting conformation derived from the crystal structure. The observations show that the bound-state conformation of α7 observed in the crystal is marginally stable, and probably reflects its participation in lattice contacts in the crystal of the bound from.
To probe whether the movement of helix α7 was coupled to a shift in the lid, principal component analysis was applied to the equilibrium MD simulation of the ligand-free Osh4 (Osh4 NPT). This analysis showed a clear coupling between the shift in the lid away from the tunnel entrance with a shift in helix α7 closer to the entrance. The overall direction of the shift corresponds approximately to the shift between the crystal structures of the bound and apo states, respectively. In the steered MD, similar high mobility of α7 is seen throughout the simulation. The nature of the dynamics does not appear to be closely coupled to the progress along the coordinate of cholesterol egress or the conformation of the lid. At no point in any of these simulations does α7 move all the way to the conformation observed in the crystallized apo state, in which the lid is absent from the protein construct. In the equilibrium simulation of the apo structure of the lid-deleted Osh4 mutant (Osh4 Δ29 NPT), starting from the crystal structure of this form, α7 was relatively stable. Taken together, these observations suggest that the closed conformation of the lid promotes a meta-stable, high mobility conformation of α7, while the open conformation of the lid is compatible with a stable, lower mobility conformation. The stability of helix α7 in the apo state (Osh4 Δ29 NPT), and the coupling of its conformation to lid opening are likely to have an important regulatory significance, since the open-state conformation of α7 is the one that appears most compatible with membrane docking, as discussed below. This point would be potentially interesting to explore using engineered disulfide crosslinks to limit the mobility of α7.
One of the more surprising observations from the crystal structures of Osh4 complex was that the ε- amino groups of Lys109 and Lys336 are located only ~3.6 Å away from each other 11
. These residues are of special interest because they are critical for Osh4 function, but they are not directly involved in cholesterol binding. The short-range interaction was not observed in the apo structure. In the apo structure, these side-chains are farther from each other, and form new interactions with sulfate ions of crystallization that were not present in the bound-state structure. These sulfate ions are coplanar with the tunnel entrance. The geometry of the interactions suggests that the sulfate ions mimic a physiological interaction with the phosphate groups of membrane phospholipids. It was therefore proposed that upon interaction with the membrane this unfavorable interaction would drive apart the Lys residues and promote the open conformation necessary for cholesterol exchange. In our simulations the ε groups of these lysine residues move to about 9 Å from each other within the first 50 ps in both equilibrium and steered simulations (Osh4-cho NPT, Osh4 NPT, Osh4-cho SMD). However the main-chains of these residues take much longer to move away from each other towards the distance actually observed in the open conformation. The MD simulation data support the concept that the close juxtaposition of the Lys residues observed in the bound-state crystal structure is unstable. However, the Lys residues have ample freedom to move apart very early in the simulation, and it does not appear that this movement alone is a driving force for lid opening. As a possible functional role, these residues could facilitate the docking of the tunnel rim area to acidic phospholipid membranes for cholesterol exchange.
Cholesterol transfer dynamics have been examined by similar steered molecular dynamics simulations for the START domains of StAR (steroidogenic acute regulatory protein) and MLN64 (metastatic lymph node 64). Like Osh4, cholesterol-binding START domains bind cholesterol in a tunnel that is completely inaccessible to solvent 20; 21
. One objective of the START domain dynamics study was to determine whether a wholesale conformational change into a molten globule 22
was necessary for cholesterol exchange, or whether the opening of a small region over the binding site was sufficient. The simulations showed only local changes in the conformation of the protein occurred during the release of cholesterol 23
. Steering of cholesterol out of the cavity mainly results in the transient opening of omega loop 1. The backbone RMSD of loop residues, increased to as much as 14 Å during the simulation. The remaining residues were stable with an RMSD range of 2 − 3 Å. Our results for the representative ORP Osh4 are thus similar to the previous results for START domains, suggesting that tunnel opening via localized lid displacement is a widespread mechanism for cholesterol exchange by soluble proteins.
In its physiological reaction, Osh4 exchanges cholesterol with membranes. The main limitation of this study is that the membrane was omitted. Nevertheless, some inferences about the transfer reaction can be drawn. Based on the crystal structures it is clear that the uptake from, and release to membranes of cholesterol by Osh4 requires the opening of the cholesterol-binding tunnel. The simulations described here show how this opening could occur in practice. The last and greatest energy barrier to cholesterol release to solution in the simulation occurs when van der Waals contacts between cholesterol and hydrophobic residues of Osh4's lid and tunnel rim are broken. These hydrophobic moieties are then exposed to aqueous solution. In the membrane context, these moieties would be exposed instead to interactions with membrane lipids. Thus in the membrane context, where these residues would have access to the hydrocarbons of the phospholipid tails, we expect that this final energy barrier would be greatly diminished. Rather, the largest energy barrier, and therefore the rate-limiting step, would be the opening of the lid.
In the extended equilibrium simulation of the apo conformation of full-length Osh4 (Osh4 NPT), the lid changed conformation with respect to the starting structure, but did not spontaneously open up. The spontaneous opening of the lid in aqueous solution is energetically unfavorable and therefore a rare event, consistent with the simulation results. In a recent study an ALPS (ArpGAP1 lipid packing sensor) motif was identified within the lid of Osh4 24
. The ALPS motif in Osh4 and in other proteins interacts preferentially with curved membranes. In our working model for the membrane exchange reaction, binding of the ALPS motif to the membrane would force the lid into the open conformation. This in turn would trigger helix α7 to move into the apo conformation, favoring stable membrane docking. It will be interesting to explore this question further with simulations of Osh4 in the presence of a phospholipid membrane.