Wojciech Kopec

Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes

Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many such molecules directly via perturbation of the plasma membrane structure and dynamics, or indirectly by modulating transmembrane protein conformational equilibria. Furthermore, for bioavailability, drugs must interact with and eventually permeate the lipid bilayer barrier on the surface of cells. Biophysical studies of the interactions of drugs and plant extracts are therefore of interest. Molecular dynamics simulations, which can access time and length scales that are not simultaneously accessible by other experimental methods, are often used to obtain quantitative molecular and thermodynamic descriptions of these interactions, often with complementary biophysical measurements. This review considers recent molecular dynamics simulations of small drug‐like molecules with membranes, and provides a biophysical description of possible routes of membrane‐mediated pharmacological effects of drugs. The review is not exhaustive, and we focus on molecules containing aromatic ring‐like structures to develop our hypotheses. We also show that some drugs and anesthetics may have an effect on the lipid bilayer analogous to that of cholesterol.

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport.

Background: Both phospholamban (PLB) and sarcolipin (SLN) regulate SERCA activity, however, only SLN uncouples SERCA. Results: The N and C termini of SLN, or the N terminus and transmembrane region of PLB, confer protein-specific function. Conclusion: SLN N terminus plays a role in dynamic interaction and uncoupling of SERCA. Significance: SERCA uncoupling by SLN increases heat production implicating SLN-SERCA interaction in muscle thermogenesis. The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is responsible for intracellular Ca2+ homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca2+-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca2+ transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues 2ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.

Molecular mechanism of Na(+),K(+)-ATPase malfunction in mutations characteristic of adrenal hypertension.

Mutations within ion-transporting proteins may severely affect their ability to traffic ions properly and thus perturb the delicate balance of ion gradients. Somatic gain-of-function mutations of the Na(+),K(+)-ATPase α1-subunit have been found in aldosterone-producing adenomas that are among the causes of hypertension. We used molecular dynamics simulations to investigate the structural consequences of these mutations, namely, Leu97 substitution by Arg (L97R), Val325 substitution by Gly (V325G), deletion of residues 93-97 (Del93-97), and deletion-substitution of residues 953-956 by Ser (EETA956S), which shows inward leak currents under physiological conditions. The first three mutations affect the structural context of the key ion-binding residue Glu327 at binding site II, which leads to the loss of the ability to bind ions correctly and to occlude the pump. The mutated residue in L97R is more hydrated, which ultimately leads to the observed proton leak. V325G mimics the structural behavior of L97R; however, it does not promote the hydration of surrounding residues. In Del93-97, a broader opening is observed because of the rearrangement of the kinked transmembrane helix 1, M1, which may explain the sodium leak measured with the mutant. The last mutant, EETA956S, opens an additional water pathway near the C-terminus, affecting the III sodium-specific binding site. The results are in excellent agreement with recent electrophysiology measurements and suggest how three mutations prevent the occlusion of the Na(+),K(+)-ATPase, with the possibility of transforming the pump into a passive ion channel, whereas the fourth mutation provides insight into the sodium binding in the E1 state.

Tuning of the Na,K-ATPase by the beta subunit.

The vital gradients of Na+ and K+ across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na+ and K+ gradients.

K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase

The H+, K+-ATPase (HKA) uses ATP to pump protons into the gastric lumen against a million-fold proton concentration gradient while counter-transporting K+ from the lumen. The mechanism of release of a proton into a highly acidic stomach environment, and the subsequent binding of a K+ ion necessitates a network of protonable residues and dynamically changing protonation states in the cation binding pocket dominated by five acidic amino acid residues E343, E795, E820, D824, and D942. We perform molecular dynamics simulations of spontaneous K+ binding to all possible protonation combinations of the acidic amino acids and carry out free energy calculations to determine the optimal protonation state of the luminal-open E2P state of the pump which is ready to bind luminal K+. A dynamic pKa correlation analysis reveals the likelihood of proton transfer events within the cation binding pocket. In agreement with in-vitro measurements, we find that E795 is likely to be protonated, and that E820 is at the center of the proton transfer network in the luminal-open E2P state. The acidic residues D942 and D824 are likely to remain protonated, and the proton redistribution occurs predominantly amongst the glutamate residues exposed to the lumen. The analysis also shows that a lower number of K+ ions bind at lower pH, modeled by a higher number of protons in the cation binding pocket, in agreement with the ‘transport stoichiometry variation’ hypothesis.

Reinforcing the membrane-mediated mechanism of action of the anti-tuberculosis candidate drug thioridazine with molecular simulations

Thioridazine is a well-known dopamine-antagonist drug with a wide range of pharmacological properties ranging from neuroleptic to antimicrobial and even anticancer activity. Thioridazine is a critical component of a promising multi-drug therapy against M. tuberculosis. Amongst the various proposed mechanisms of action, the cell membrane-mediated one is peculiarly tempting due to the distinctive feature of phenothiazine drug family to accumulate in selected body tissues. In this study, we employ long-scale molecular dynamics simulations to investigate the interactions of three different concentrations of thioridazine with zwitterionic and negatively charged model lipid membranes. Thioridazine partitions into the interfacial region of membranes and modifies their structural and dynamic properties, however dissimilarly so at the highest membrane-occurring concentration, that appears to be obtainable only for the negatively charged bilayer. We show that the origin of such changes is the drug induced decrease of the interfacial tension, which ultimately leads to the significant membrane expansion. Our findings support the hypothesis that the phenothiazines therapeutic activity may arise from the drug–membrane interactions, and reinforce the wider, emerging view of action of many small, bioactive compounds.

Conformations of double-headed, triple-tailed phospholipid oxidation lipid products in model membranes.

Products of phospholipid oxidation can produce lipids with a carbonyl moiety at the end of a shortened lipid acyl tail, such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The carbonyl tail of POVPC can covalently bond to the free tertiary amine of a phosphatidylethanolamine lipid in a Schiff base reaction to form a conjugate lipid (SCH) with two head groups, and three acyl tails. We investigate the conformations and properties of this unique class of adduct lipids using molecular dynamics simulations, and show that their insertion into lipid bilayers of POPC increases the average cross-sectional area per lipid and decreases bilayer thickness. Significant increase in acyl tail fluidity is only observed at 25% SCH concentration. The SCH occupies a larger area per lipid than expected for a lipid with three acyl tails, owing to the interfacial location of the long spacer between the two head groups of the SCH. Schiff base formation of lipids can alter the concentration, homeostasis and localizations of phosphatidylserine and phosphatidylethanol lipids in membranes, and can therefore influence several membrane-associated processes including fusion and budding. The current work provides the first detailed structural model of this unique new class of lipids that may have important roles to play in modulating membrane properties and cell physiology.

K+ Congeners That Do Not Compromise Na+ Activation of the Na+,K+-ATPase HYDRATION OF THE ION BINDING CAVITY LIKELY CONTROLS ION SELECTIVITY

Background: The Na+,K+-ATPase discriminates between similar and abundant ions. Results: The K+ congener acetamidinium interacts with the outward facing sites of Na+,K+-ATPase, but does not interact with the inward facing sites. Conclusion: Water in the ion binding cavity regulates ion selectivity of the Na+,K+-ATPase. Significance: This study identifies new determinants of ion selectivity of K+-transporting P-type pumps. The Na+,K+-ATPase is essential for ionic homeostasis in animal cells. The dephosphoenzyme contains Na+ selective inward facing sites, whereas the phosphoenzyme contains K+ selective outward facing sites. Under normal physiological conditions, K+ inhibits cytoplasmic Na+ activation of the enzyme. Acetamidinium (Acet+) and formamidinium (Form+) have been shown to permeate the pump through the outward facing sites. Here, we show that these cations, unlike K+, are unable to enter the inward facing sites in the dephosphorylated enzyme. Consistently, the organic cations exhibited little to no antagonism to cytoplasmic Na+ activation. Na+,K+-ATPase structures revealed a previously undescribed rotamer transition of the hydroxymethyl side chain of the absolutely conserved Thr772 of the α-subunit. The side chain contributes its hydroxyl to Na+ in site I in the E1 form and rotates to contribute its methyl group toward K+ in the E2 form. Molecular dynamics simulations to the E1·AlF4−·ADP·3Na+ structure indicated that 1) bound organic cations differentially distorted the ion binding sites, 2) the hydroxymethyl of Thr772 rotates to stabilize bound Form+ through water molecules, and 3) the rotamer transition is mediated by water traffic into the ion binding cavity. Accordingly, dehydration induced by osmotic stress enhanced the interaction of the congeners with the outward facing sites and profoundly modified the organization of membrane domains of the α-subunit. These results assign a catalytic role for water in pump function, and shed light on a backbone-independent but a conformation-dependent switch between H-bond and dispersion contact as part of the catalytic mechanism of the Na+,K+-ATPase.

Accelerating All-Atom MD Simulations of Lipids Using a Modified Virtual-Sites Technique.

We present two new implementations of the virtual sites technique which completely suppresses the degrees of freedom of the hydrogen atoms in a lipid bilayer allowing for an increased time step of 5 fs in all-atom simulations of the CHARMM36 force field. One of our approaches uses the derivation of the virtual sites used in GROMACS while the other uses a new definition of the virtual sites of the CH2 groups. Our methods is tested on a DPPC (no unsaturated chain), a POPC (one unsaturated chain), and a DOPC (two unsaturated chains) lipid bilayers. We calculate various physical properties of the membrane of our simulations with and without virtual sites and explain the differences and similarity observed. The best agreements are obtained for the GROMACS original virtual sites on the DOPC bilayer where we get an area per lipid of 67.3 ± 0.3 Å(2) without virtual sites and 67.6 ± 0.3 Å(2) with virtual sites. In conclusion the virtual-sites technique on lipid membranes is a powerful simulation tool, but it should be used with care. The procedure can be applied to other force fields and lipids in a straightforward manner.

Insights into the role of cyclic ladderane lipids in bacteria from computer simulations

Ladderanes, which are multiple fused cyclobutane rings, are unique structures available only in nature. Anammox bacteria produce ladderane phospholipids during their life cycle, but the synthesis mechanism still remains a mystery. The function of ladderane lipids in the membrane is unclear as well. According to previous speculations, ladderane moieties of the bilayer might decrease permeability for certain molecules, which should not diffuse out of the compartment enclosed by the ladderane-containing membrane. We report the first atomistic-precision molecular dynamics simulations of bilayers containing ladderane lipids. The structural and thermodynamics differences among (1) pure ladderane containing bilayer, (2) POPC bilayer, and (3) their equimolar mixture are discussed. Potentials of mean force are reported for the translocation of a hydrazine molecule through all investigated bilayers. All bilayers offer a potential energy barrier to hydrazine. Contrary to expectations, the presence of the ladderane lipids somewhat lowers the barrier for translocation of hydrazine. We conclude that the presence of ladderane phospholipids in anammox bacteria does not serve as a barrier to hydrazine. It may serve as a barrier to larger and noxious intermediates in the anammox reaction, or, the true mission of ladderane lipids must be located in a different plane.