Drastic differences between the release kinetics of two highly related porphyrins in liposomal membranes: mTHPP and pTHPP.

HYPOTHESIS: The release of hydrophobic compounds from liposomal membranes occurs by partitioning and is thus determined by the physicochemical properties (e.g. logP and water solubility) of the drug. We postulate that even minor structural differences, e.g. the position of the phenolic OH-group of the hydrophobic porphyrins mTHPP and pTHPP (meta vs. para substitution), distinctly affect their partitioning and release behavior from liposomes. EXPERIMENTS: The release and redistribution of mTHPP and pTHPP from lecithin or POPC/POPG liposomes to different acceptor particles (DSPE-mPEG micelles and liposomes) was studied by asymmetrical flow field-flow fractionation to separate donor and acceptor particles. Reversed phase HPLC was applied to detect differences in partitioning. Molecular dynamics (MD) simulations were carried out to obtain molecular insight in the different behavior of the two compounds inside a lipid bilayer. FINDINGS: Despite the minor differences in chemical structure, mTHPP is more hydrophobic and redistributes much slower to both acceptor phases than pTHPP. MD simulations indicate that compared to pTHPP, mTHPP makes stronger hydrogen bonds with the lipid head groups, is oriented more parallel to the lipid tails and is embedded slightly deeper in the membrane.

Engineering a membrane-binding protein to trimerize and induce high membrane curvature.

The annexins are a family of Ca2+-dependent peripheral membrane proteins. Several annexins are implicated in plasma membrane repair and are overexpressed in cancer cells. Annexin A4 (ANXA4) and annexin A5 (ANXA5) form trimers that induce high curvature on a membrane surface, a phenomenon deemed to accelerate membrane repair. Despite being highly homologous to ANXA4, annexin A3 (ANXA3) does not form trimers on the membrane surface. Using molecular dynamics simulations, we have reverse engineered an ANXA3-mutant to trimerize on the surface of the membrane and induce high curvature reminiscent of ANXA4. In addition, atomic force microscopy images show that, like ANXA4, the engineered protein forms crystalline arrays on a supported lipid membrane. Despite the trimer-forming and curvature-inducing properties of the engineered ANXA3, it does not accumulate near a membrane lesion in laser-punctured cells and is unable to repair the lesion. Our investigation provides insights into the factors that drive annexin-mediated membrane repair and shows that the membrane-repairing property of trimer-forming annexins also necessitates high membrane binding affinity, other than trimer formation and induction of negative membrane curvature.

The molecular basis of the antidepressant action of the magic mushroom extract, psilocin.

Magic mushrooms, and their extract psilocybin, are well-known for their psychedelic properties and recreational use. Psilocin, the bio-active form of psilocybin, can potentially treat various psychiatric diseases. Psilocin putatively exerts its psychedelic effect as an agonist to the serotonin 2A receptor (5-HT2AR), which is also the receptor for the neurological hormone serotonin. The two key chemical differences between the two molecules are first, that the primary amine in serotonin is replaced with a tertiary amine in psilocin, and second, the hydroxyl group is substituted differently on the aromatic ring. Here, we find that psilocin can bind to 5-HT2AR with an affinity higher than serotonin, and provide the molecular logic behind the higher binding affinity of psilocin using extensive molecular dynamics simulations and free energy calculations. The binding free energy of psilocin is dependent upon the protonation states of the ligands, as well as that of the key residue in the binding site: Aspartate 155. We find that the tertiary amine of psilocin, and not the altered substitution of the hydroxyl group in the ring is responsible for the increased affinity of psilocin. We propose design rules for effective antidepressants based on molecular insights from our simulations.

Interaction of psychedelic tryptamine derivatives with a lipid bilayer.

Naturally occurring psychedelics have been used for a long time as remedies or in religious ceremonies and recreational activities. Recent studies have proven the therapeutic potential of some psychedelic compounds to safely treat a wide range of diseases such as anxiety, depression, migraine, and addiction. It is hypothesized that psychedelic compounds like tryptamines can exert their effects by two possible mechanisms: binding to the transmembrane serotonin receptor and/or modifying the properties of the neuronal membrane that can alter the conformational equilibrium and desensitize receptors. The impact of three different tryptamine class compounds with a tertiary amine (dimethyltryptamine, bufotenine, and 5-MeO-DMT) in both neutral and charged forms on a model bilayer lipid membrane are studied using all-atom MD simulations. All compounds partition into the bilayer, and change membrane properties, but to different extents. We determine the tendency of compounds to partition into the membrane by free energy calculations. Neutral tryptamines partition into the bilayer almost completely. Dimethyltryptamine and 5-MeO-DMT cross the membrane spontaneously during the simulation time, but bufotenine does not, although it has the maximum effect on the structural properties of the membrane. However, protonated compounds partition partially into the bilayer and cannot pass through the middle of the membrane during the simulation time. In this way, subtle alteration of chemical structure can play a significant role in the improvement or deterioration of partitioning of these compounds into the bilayer and their passage across the membrane.

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles.

The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.