Comparative study of the AT1 receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers

Drug–membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution ¹H and ¹³C nuclear magnetic resonance (NMR) and solid state ¹³C and ³¹P (NMR) spectroscopies. In addition, ³¹P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT₁ receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.     

The application of solid-state NMR spectroscopy to study candesartan cilexetil (TCV-116) membrane interactions. Comparative study with the AT1R antagonist drug olmesartan

ΑΤ1 receptor (AT1R) antagonists exert their antihypertensive effects by preventing the vasoconstrictive hormone AngII to bind to the AT1 receptor. It has been proposed that these biological effects are mediated through a two-step mechanism reaction. In the first step, they are incorporated in the core of the lipid bilayers and in the second step they reach the active site of the receptor through lateral diffusion. In this model, drug/membrane interactions are key elements for the drugs achieving inhibition at the AT1 receptor. In this work, the interactions of the prodrug candesartan cilexetil (TCV-116) with lipid bilayers are studied at molecular detail. Solid-state ¹³C-CP/MAS, 2D ¹H-¹H NOESY NMR spectroscopy and in silico calculations are used. TCV-116 and olmesartan, another drug which acts as an AT1R antagonist are compared for their dynamic effects in lipid bilayers using solid-state ²H-NMR. We find a similar localization of TCV-116 compared to other AT1 antagonists in the intermediate polar region. In addition, we can identify specific local interactions. These interactions may be associated in part with the discrete pharmacological profiles observed for different antagonists.     

Determination of candesartan cilexetil, candesartan and a metabolite in human plasma and urine by liquid chromatography and fluorometric detection

Liquid chromatographic methods are described for the determination of a new effective anti-hypertensive drug candesartan (CV-11974), its prodrug candesartan cilexetil (TCV-116) and a metabolite, CV-15959 in human plasma and urine. The assays comprise liquid–liquid extraction and separation on a phenyl column with fluorometric detection. The methods give absolute recoveries of 70, 83 and 78% for candesartan cilexetil, candesartan and CV-15959, respectively, and the limit of quantification is 5, 1 and 3 nM of plasma (RSD<20%), respectively. The methods were applied to plasma and urine samples from biopharmaceutical and clinical studies in man.     

Solid-state NMR and MD simulations of the antiviral drug amantadine solubilized in DMPC bilayers

The interactions of ¹⁵N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of ³¹P chemical shielding and ¹⁴N quadrupolar interactions and by the MD simulation.     

Effects of a carane derivative local anesthetic on a phospholipid bilayer studied by molecular dynamics simulation

Molecular dynamics (MD) simulations of two hydrated palmitoyloleoylphosphatidylcholine (POPC) bilayers each containing eight carane derivative (KP-23) local anesthetic (LA) molecules in neutral (POPC-LA) or protonated (POPC-LAH) forms were carried out to investigate the effect of KP-23 and its protonation on the bilayer. 3-ns trajectories were used for analyses. A pure POPC bilayer was employed as a reference system. In both POPC-LA and POPC-LAH systems a few KP-23 molecules intercalated into the bilayer and moved near the bilayer/water interface. They were located on the hydrophobic core side of the interface in the POPC-LA bilayer, but on the water phase side in the POPC-LAH bilayer. The order of the POPC chains was higher in the POPC-LA bilayer than in the pure POPC bilayer and was lower in the POPC-LAH bilayer. Interactions between polar groups of KP-23 and POPC or water were responsible for a lower hydration of POPC headgroups in POPC bilayers containing KP-23 than in the pure POPC bilayer. KP-23 molecules were found to form aggregates both in POPC-LA and POPC-LAH bilayers. Due to higher amphiphilicity of LAH, the LAH aggregate was more micelle-like and larger than the LA one. The results demonstrate the rapid timescales of the initial processes that take place at and near the bilayer interface as well as details of the atomic level interactions between local anesthetic and the lipid matrix of a cell membrane.     

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.     

Molecular dynamics simulation study of interaction between a class IIa bacteriocin and its immunity protein

Molecular dynamics (MD) simulations of carnobacteriocin B2 (CbnB2), a structurally well-characterized class IIa bacteriocin, and its immunity protein (ImB2) in lipid bilayer environment have been conducted to explore the interaction between them. Six 30-ns simulations were conducted in DPPC or POPG bilayer systems. In these simulations, ImB2 was placed in the aqueous layer with different orientations facing CbnB2 to sample all the faces of ImB2. The MD results indicate that (i) while CbnB2 remained embedded in the bilayer, it tends to move toward the interface, and (ii) the presence of CbnB2 in the DPPC bilayer attracts ImB2 toward the bilayer. In one of the orientations in DPPC bilayer system (simulation 1), ImB2 penetrates the bilayer and interacts with CbnB2 by ion-pair interaction. At several instances toward the later half of the simulation (15-30 ns), ImB2 and CbnB2 were found to form salt-bridge between Arg95 of ImB2 and Glu24 of CbnB2. Simulation in POPG bilayer displayed strong interaction between the positively charged ImB2 and the negatively charged polar head groups of the POPG molecules at the lipid-water interface. However, ImB2 was not able to penetrate the bilayer thereby preventing any interaction between ImB2 and CbnB2.     

Comparative study of the interaction of fullerenol nanoparticles with eukaryotic and bacterial model membranes using solid-state NMR and FTIR spectroscopy

Native fullerene is notoriously insoluble in water and forms aggregates toxic to cell membranes, thus limiting its use in nanomedicine. In contrast, water-soluble fullerenol is compatible with biological systems and shows low in vivo toxicity on human cell lines. The interaction mechanism between these hydrophilic nanoparticles and biological membranes is however not well understood. Therefore, in this work, the effect of fullerenol on model eukaryotic and bacterial membranes was investigated using (31)P- and (2)H solid-state NMR as well as FTIR spectroscopy. DPPC/cholesterol and DPPC/DPPG bilayers were used to mimic eukaryotic and bacterial cell membranes, respectively. Our results show low affinity of fullerenol for DPPC/cholesterol bilayers but a clear interaction with model bacterial membranes. A preferential affinity of fullerenol for the anionic phospholipids DPPG in DPPC/DPPG membranes is also observed. Our data suggest that fullerenol remains at the water/bilayer interface of eukaryote-like membranes. They also indicate that the presence of a polar group such as DPPG's hydroxyl moiety at the bilayer surface plays a key role in the interaction of fullerenol with membranes. Hydrogen bonding of fullerenol nanoparticles with DPPGs' OH groups is most likely responsible for inducing lipid segregation in the lipid bilayer. Moreover, the location of the nanoparticles in the polar region of DPPG-rich regions appears to disturb the acyl chain packing and increase the membrane fluidity. The preferential interaction of fullerenol with lipids mostly found in bacterial membranes is of great interest for the design of new antibiotics.     

Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37

LL-37 is a cationic, amphipathic alpha-helical antimicrobial peptide found in humans that kills cells by disrupting the cell membrane. To disrupt membranes, antimicrobial peptides such as LL-37 must alter the hydrophobic core of the bilayer. Differential scanning calorimetry and deuterium ((2)H) NMR experiments on acyl chain perdeuterated lipids demonstrate that LL-37 inserts into the hydrophobic region of the bilayer and alters the chain packing and cooperativity. The results show that hydrophobic interactions between LL-37 and the hydrophobic acyl chains are as important for the ability of this peptide to disrupt lipid bilayers as its electrostatic interactions with the polar headgroups. The (2)H NMR data are consistent with the previously determined surface orientation of LL-37 (Henzler Wildman, K. A., et al. (2003) Biochemistry 42, 6545) with an estimated 5-6 A depth of penetration of the hydrophobic face of the amphipathic helix into the hydrophobic interior of the bilayer. LL-37 also alters the material properties of lipid bilayers, including the area per lipid, hydrophobic thickness, and coefficient of thermal expansion in a manner that varies with lipid type and temperature. Comparison of the effect of LL-37 on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC-d(31)) and 1,2-dimyristoyl-phosphatidylcholine (DMPC-d(54)) at different temperatures demonstrates the importance of bilayer order in determining the type and extent of disordering and disruption of the hydrophobic core by LL-37. One possible explanation, which accounts for both the (2)H NMR data presented here and the known surface orientation of LL-37 under identical conditions, is that bilayer order influences the depth of insertion of LL-37 into the hydrophobic/hydrophilic interface of the bilayer, altering the balance of electrostatic and hydrophobic interactions between the peptide and the lipids.     

Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers

The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.     

Vstx1, a modifier of Kv channel gating, localizes to the interfacial region of lipid bilayers

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.     

Fusogenic Alzheimer's peptide fragment Abeta (29-42) in interaction with lipid bilayers: secondary structure, dynamics, and specific interaction with phosphatidyl ethanolamine polar heads as revealed by solid-state NMR

The interaction of the native Alzheimer's peptide C-terminal fragment Abeta (29-42), and two mutants (G33A and G37A) with neutral lipid bilayers made of POPC and POPE in a 9:1 molar ratio was investigated by solid-state NMR. This fragment and the lipid composition were selected because they represent the minimum requirement for the fusogenic activity of the Alzheimer's peptide. The chemical shifts of alanine methyl isotropic carbon were determined by MAS NMR, and they clearly demonstrated that the major form of the peptide equilibrated in membrane is not in a helical conformation. (2)H NMR, performed with acyl chain deuterated POPC, demonstrated that there is no perturbation of the acyl chain's dynamics and of the lipid phase transition temperature. (2)H NMR, performed with alanine methyl-deuterated peptide demonstrated that the peptide itself has a limited mobility below and above the lipid phase transition temperature (molecular order parameter equal to 0.94). MAS (31)P NMR revealed a specific interaction with POPE polar head as seen by the enhancement of POPE phosphorus nuclei T(2) relaxation. All these results are in favor of a beta-sheet oligomeric association of the peptide at the bilayer interface, preferentially recruiting phosphatidyl ethanolamine polar heads.     

Solid-state NMR studies of a diverged microsomal amino-proximate delta12 desaturase peptide reveal causes of stability in bilayer: tyrosine anchoring and arginine snorkeling

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal delta12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L(alpha) phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8 degrees with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580 ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT₁ receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartan's pathway towards the AT₁ receptor.     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Cholesterol Binds in a Reversed Orientation to TCRβ-TM in Which Its OH Group is Localized to the Center of the Lipid Bilayer

T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRβ subunit (TCRβ-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRβ. However, the atomic interaction of cholesterol or CS with TCRβ remains elusive. Here, we determined the cholesterol and CS binding site of TCRβ-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRβ-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRβ-TM, validating the NMR and MD results.     

Effects of phospholipid unsaturation on the membrane/water interface: a molecular simulation study.

Molecular dynamics (MD) simulations of fully hydrated bilayers in the liquid-crystalline state made of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) or 1-palmitoyl-2-elaidoyl-phosphatidylcholine (PEPC) were carried out to investigate the effect of the incorporation of a double bond in the phosphatidylcholine (PC) beta-chain (cis or trans) on the membrane/water interface. The bilayers reached thermal equilibrium after 3 and 1 ns of MD simulations, respectively, and productive runs were carried out for 3 ns for each bilayer. As reference systems, the 1,2-dimyristoyl-phosphatidylcholine (DMPC) bilayer (M. Pasenkiewicz-Gierula, Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi, 1999, Biophys. J. 76:1228-1240) and DMPC-cholesterol (Chol) bilayer containing 22 mol % Chol (M. Pasenkiewicz-Gierula, T. Róg, K. Kitamura, A. and Kusumi, 2000, Biophys. J. 78:1376-1389) were used. The study shows that at the interface of POPC, PEPC, and DMPC-Chol bilayers, average numbers of PC-water and PC-PC interactions are similar and, respectively, greater and smaller than in the DMPC bilayer. The average area/PC in mono-unsaturated bilayers is approximately 4 A(2) larger than in the DMPC bilayer; nevertheless, a strong correlation was found between a single molecular area (SMA) of a PC and the number of interactions this PC makes; i.e., PCs (either saturated or unsaturated) with the same SMA form similar numbers of intermolecular links. The numbers and corresponding SMAs are distributed about averages pertinent to each bilayer. No significant difference between cis and trans bonds was found.