Computational Insights into the Role of Cholesterol in Inverted Hexagonal Phase Stabilization and Endosomal Drug Release.

Cholesterol is a major component of many lipid-based drug delivery systems, including cationic lipid nanoparticles. Despite its critical role in the drug release stage, the underlying molecular mechanism by which cholesterol assists in endosomal escape remains unclear. An efficient drug release from the endosome requires endosomal disruption. This disruption is believed to involve a lamellar-to-inverted hexagonal (Lα-HII) phase transition upon fusion of the lipid nanoparticle with the endosomal membrane. We used molecular dynamics simulations to study the structural properties of HII systems composed of an anionic lipid distearoyl phosphatidylserine (DSPS), an ionizable cationic lipid (KC2H), and cholesterol for several hydration levels and molar ratios. This system corresponds to the lipid mixtures in the hypothesized HII structure formed upon fusion and is of interest for the rational design of ionizable cationic lipids, including KC2, for an optimal drug release. Simulations suggest a geometry- and symmetry-driven lipid sorting and cholesterol-DSPS co-location around the water cores. Cholesterol preferentially co-locates with negatively charged saturated DSPS lipids at interstitial angles. The observed cholesterol-DSPS co-location results in an overall increase in the DSPS acyl chains' order parameters, which we propose to assist in stabilizing the HII phase by stretching the DSPS acyl chains for filling the voids formed by three adjacent lipid tubules. Furthermore, a systematic increase in the cholesterol concentration increased the lattice plane spacing and the water core radius but decreased the undulations along the lipid tubule axis. We propose that cholesterol and the degree of saturation/polyunsaturation of the lipid acyl chains, and not the lipid charge, are the main contributors in facilitating the Lα-HII phase transition and stabilizing/destabilizing the formed HII phase, whereas the positive charge of the ionizable cationic lipid promotes the LNP-endosomal membrane adhesion and assists in initiating the fusion process at the local contact area. We also propose that the effect of cholesterol on the HII structure and curvature is the main underlying reason for the well-documented HII stabilization and destabilization at low and high molar concentrations of cholesterol, respectively.

An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair.

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys122 The impacts of the AI helix on active-site dynamics and positioning of Lys122 are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface.