Aminoadamantane drugs are lipophilic amines that block the membrane-embedded influenza A M2 WT (wild type) ion channel protein. The comparative effects of amantadine (Amt) and its synthetic spiro[pyrrolidine-2,2′-adamantane] (AK13) analogue in dimyristoylphosphatidylcholine (DMPC) bilayers were studied using a combination of experimental biophysical methods, differential scanning calorimetry (DSC), X-ray diffraction, solid-state NMR (ssNMR) spectroscopy, and molecular dynamics (MD) simulations. All three experimental methods pointed out that the two analogues perturbed drastically the DMPC bilayers with AK13 to be more effective at high concentrations. AK13 was tolerated in lipid bilayers at very high concentrations, while Amt was crystallized. This is an important consideration in the formulations of drugs as it designates a limitation of Amt incorporation. MD simulations verify provided details about the strong interactions of the drugs in the interface region between phosphoglycerol backbone and lipophilic segments. The two drugs form hydrogen bonding with both water and sn-2 carbonyls in their amine form or water and phosphate oxygens in their ammonium form. Such localization of the drugs explains the DMPC bilayers reorientation and their strong perturbing effect evidenced by all biophysical methodologies applied.
Ras proteins are membrane-anchored
GTPases that regulate key cellular
signaling networks. It has been recently shown that different anionic
lipid types can affect the properties of Ras in terms of dimerization/clustering
on the cell membrane. To understand the effects of anionic lipids
on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom
molecular dynamics simulations of the dimer K-Ras4B in the presence
and absence of Raf[RBD/CRD] effectors on two model anionic lipid membranes:
one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another
one with enhanced concentration of anionic lipids containing 50% mol
DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils
the orientational space of dimeric K-Ras4B and shows that the stability
of the dimer is enhanced on the membrane containing a high concentration
of anionic lipids in the absence of Raf effectors. This enhanced stability
is also observed in the presence of Raf[RBD/CRD] effectors although
it is not influenced by the concentration of anionic lipids in the
membrane, but rather on the ability of Raf[CRD] to anchor to the membrane.
We generate dominant K-Ras4B conformations by Markov state modeling
and yield the population of states according to the K-Ras4B orientation
on the membrane. For the membrane containing anionic lipids, we observe
correlations between the diffusion of K-Ras4B and PIP2 and anchoring
of anionic lipids to the Raf[CRD] domain. We conclude that the presence
of effectors with the Raf[CRD] domain anchoring on the membrane as
well as the membrane composition both influence the conformational
stability of the K-Ras4B dimer, enabling the preservation of crucial
interface interactions.
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