SGTx1, a Kv channel gating-modifier toxin, binds to the interfacial region of lipid bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.     

SGTx1, a Kv Channel Gating-Modifier Toxin, Binds to the Interfacial Region of Lipid Bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.     

Interactions Between a Voltage Sensor and a Toxin via Multiscale Simulations

Gating-modifier toxins inhibit voltage-gated ion channels by binding the voltage sensors (VS) and altering the energetics of voltage-dependent gating. These toxins are thought to gain access to the VS via the membrane (i.e., by partitioning from water into the membrane before binding the VS). We used serial multiscale molecular-dynamics (MD) simulations, via a combination of coarse-grained (CG) and atomistic (AT) simulations, to study how the toxin VSTx1, which inhibits the archeabacterial voltage-gated potassium channel KvAP, interacts with an isolated membrane-embedded VS domain. In the CG simulations, VSTx1, which was initially located in water, partitioned into the headgroup/water interface of the lipid bilayer before binding the VS. The CG configurations were used to generate AT representations of the system, which were subjected to AT-MD to further evaluate the stability of the complex and refine the predicted VS/toxin interface. VSTx1 interacted with a binding site on the VS formed by the C-terminus of S1, the S1-S2 linker, and the N-terminus of S4. The predicted VS/toxin interactions are suggestive of toxin-mediated perturbations of the interaction between the VS and the pore domain of Kv channels, and of the membrane. Our simulations support a membrane-access mechanism of inhibition of Kv channels by VS toxins. Overall, the results show that serial multiscale MD simulations may be used to model a two-stage process of protein-bilayer and protein-protein interactions within a membrane.     

Peptide-lipid interactions of the beta-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a beta-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. (31)P and (2)H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other beta-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

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.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

Lipid bilayer deformation and the free energy of interaction of a Kv channel gating-modifier toxin

A number of membrane proteins act via binding at the water/lipid bilayer interface. An important example of such proteins is provided by the gating-modifier toxins that act on voltage-gated potassium (Kv) channels. They are thought to partition to the headgroup region of lipid bilayers, and so provide a good system for probing the nature of interactions of a protein with the water/bilayer interface. We used coarse-grained molecular dynamics simulations to compute the one-dimensional potential of mean force (i.e., free energy) profile that governs the interaction between a Kv channel gating-modifier toxin (VSTx1) and model phospholipid bilayers. The reaction coordinate sampled corresponds to the position of the toxin along the bilayer normal. The course-grained representation of the protein and lipids enabled us to explore extended time periods, revealing aspects of toxin/bilayer dynamics and energetics that would be difficult to observe on the timescales currently afforded by atomistic molecular dynamics simulations. In particular, we show for this model system that the bilayer deforms as it interacts with the toxin, and that such deformations perturb the free energy profile. Bilayer deformation therefore adds an additional layer of complexity to be addressed in investigations of membrane/protein systems. In particular, one should allow for local deformations that may arise due to the spatial array of charged and hydrophobic elements of an interfacially located membrane protein.     

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 ³¹P 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. ²H 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. ³¹P 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, ³¹P and ²H 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.     

Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. ³¹P and ²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Comparative molecular dynamics simulations of the antimicrobial peptide CM15 in model lipid bilayers

We report altogether 3-μs molecular dynamics (MD) simulations of the antimicrobial peptide CM15 to systematically investigate its interaction with two model lipid bilayers, pure POPC and mixed POPG:POPC (1:2). Starting with either an α-helical or a random-coil conformation, CM15 is found to insert into both bilayers. Peptide-lipid interaction is stronger with the anionic POPG:POPC than the zwitterionic POPC, which is largely attributed to the electrostatic attraction between CM15 and the negatively charged POPG. Simulations initiated with CM15 as a random coil allowed us to study peptide folding at the lipid-water interface. Interestingly, CM15 folding appears to be faster in POPC than POPG:POPC, which may be explained by a lower activation energy barrier of structural rearrangement in the former system. Our data also suggest that compared with the random-coil conformation, CM15 in a pre-folded α-helix has significantly reduced interactions with the lipids, indicating that peptide initial structures may bias the simulation results considerably on the 100-ns timescale. The implications of this result should be considered when preparing and interpreting future AMP simulations.     

Interaction of protegrin-1 with lipid bilayers: membrane thinning effect

Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer.     

Membrane composition determines pardaxin's mechanism of lipid bilayer disruption

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.     

A coarse-grained approach to studying the interactions of the antimicrobial peptides aurein 1.2 and maculatin 1.1 with POPG/POPE lipid mixtures

In the present work we investigated the differential interactions of the antimicrobial peptides (AMPs) aurein 1.2 and maculatin 1.1 with a bilayer composed of a mixture of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). We carried out molecular dynamics (MD) simulations using a coarse-grained approach within the MARTINI force field. The POPE/POPG mixture was used as a simple model of a bacterial (prokaryotic cell) membrane. The results were compared with our previous findings for structures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a representative lipid of mammalian cells. We started the simulations of the peptide–lipid system from two different initial conditions: peptides in water and peptides inside the hydrophobic core of the membrane, employing a pre-assembled lipid bilayer in both cases. Our results show similarities and differences regarding the molecular behavior of the peptides in POPE/POPG in comparison to their behavior in a POPC membrane. For instance, aurein 1.2 molecules can adopt similar pore-like structures on both POPG/POPE and POPC membranes, but the peptides are found deeper in the hydrophobic core in the former. Maculatin 1.1 molecules, in turn, achieve very similar structures in both kinds of bilayers: they have a strong tendency to form clusters and induce curvature. Therefore, the results of this study provide insight into the mechanisms of action of these two peptides in membrane leakage, which allows organisms to protect themselves against potentially harmful bacteria.

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Graphical Abstract Aurein pore structure (green) in a lipid bilayer composed by POPE (blue) and POPG (red) mixture. It is possible to see water beads (light blue) inside the pore.

     

Interfacial tryptophan residues: a role for the cation-pi effect?

Integral membrane proteins are characterized by having a preference for aromatic residues, e.g., tryptophan (W), at the interface between the lipid bilayer core and the aqueous phase. The reason for this is not clear, but it seems that the preference is related to a complex interplay between steric and electrostatic forces. The flat rigid paddle-like structure of tryptophan, associated with a quadrupolar moment (aromaticity) arising from the pi-electron cloud of the indole, interacts primarily with moieties in the lipid headgroup region hardly penetrating into the bilayer core. We have studied the interaction between the nitrogen moiety of lipid molecule headgroups and the pi-electron distribution of gramicidin (gA) tryptophan residues (W9, W11, W13, and W15) using molecular dynamics (MD) simulations of gA embedded in two hydrated lipid bilayers composed of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoylphosphatidyl-choline (POPC), respectively. We use a force field model for tryptophan in which polarizability is only implicit, but we believe that classical molecular dynamics force fields are sufficient to capture the most prominent features of the cation-pi interaction. Our criteria for cation-pi interactions are based on distance and angular requirements, and the results from our model suggest that cation-pi interactions are relevant for W(PE)1), W(PE)13, W(PE)15, and, to some extent, W(PC)11 and W(PC)13. In our model, W9 does not seem to engage in cation-pi interactions with lipids, neither in POPE nor POPC. The criteria for the cation-pi effect are satisfied more often in POPE than in POPC, whereas the H-bonding ability between the indole donor and the carbonyl acceptor is similar in POPE and POPC. This suggests an increased affinity for lipids with ethanolamine headgroups to transmembrane proteins enriched in interfacial tryptophans.     

Insertion of Dengue E into lipid bilayers studied by neutron reflectivity and molecular dynamics simulations

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.     

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.     

Solution structure and lipid membrane partitioning of VSTx1, an inhibitor of the KvAP potassium channel

VSTx1 is a voltage sensor toxin from the spider Grammostola spatulata that inhibits KvAP, an archeabacterial voltage-activated K(+) channel whose X-ray structure has been reported. Although the receptor for VSTx1 and the mechanism of inhibition are unknown, the sequence of the toxin is related to hanatoxin (HaTx) and SGTx, two toxins that inhibit eukaryotic voltage-activated K(+) channels by binding to voltage sensors. VSTx1 has been recently shown to interact equally well with lipid membranes that contain zwitterionic or acidic phospholipids, and it has been proposed that the toxin receptor is located within a region of the channel that is submerged in the membrane. As a first step toward understanding the inhibitory mechanism of VSTx1, we determined the three-dimensional solution structure of the toxin using NMR. Although the structure of VSTx1 is similar to HaTx and SGTx in terms of molecular fold and amphipathic character, the detailed positions of hydrophobic and surrounding charged residues in VSTx1 are very different than what is seen in the other toxins. The amphipathic character of VSTx1, notably the close apposition of basic and hydrophobic residues on one face of the toxin, raises the possibility that the toxin interacts with interfacial regions of the membrane. We reinvestigated the partitioning of VSTx1 into lipid membranes and find that VSTx1 partitioning requires negatively charged phospholipids. Intrinsic tryptophan fluorescence and acrylamide quenching experiments suggest that tryptophan residues on the hydrophobic surface of VSTx1 have a diminished exposure to water when the toxin interacts with membranes. The present results suggest that if membrane partitioning is involved in the mechanism by which VSTx1 inhibits voltage-activated K(+) channels, then binding of the toxin to the channel would likely occur at the interface between the polar headgroups and the hydrophobic phase of the membrane.     

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.     

Gap junction hemichannel interactions with zwitterionic lipid, anionic lipid, and cholesterol: molecular simulation studies

Interactions with membrane lipids can exert dramatic functional consequences on gap junction proteins. Recent experimental work has highlighted the importance of anionic lipids and cholesterol in facilitating channel activity. In this work, we have employed a coarse-grained molecular model in conjunction with molecular dynamics (MD) simulations to study the interactions between a connexin 26 (Cx26) hemichannel and a number of lipid species, including palmitoyloleoylphosphatidylcholine (POPC), anionic palmitoyloleoylphosphatidic acid (POPA), and cholesterol, in order to identify sites at the protein interface which may exhibit preferential, specific binding to these lipids, as well as determine the characteristics of these interactions. We have also employed an atomistic model of Cx26 embedded in a mixed PA/PC bilayer as a comparison and to elucidate further lipid-protein interactions. Our simulation results suggest enrichment of interfacial PA at the intracellular leaflet at high bulk PA concentrations. PC can form tight binding interactions with the hemichannel, particularly at intersubunit crevices (classical nonannular sites). In mixed bilayers, however, POPA competes with POPC for these sites, displacing the latter in some cases. While the residues responsible for interactions with PC and PA are similar, the latter exhibits a unique property of being capable of forming stable hydrophilic contacts with multiple residues spanning two different adjacent subunits at both leaflets of the bilayer, as opposed to POPC which can only do so at the extracellular side. These results suggest that POPA may be essential to channel function by acting as an intersubunit lipid bridge. Additionally, we propose that the compositional enrichment of POPA at the Cx26 interface may serve important roles in voltage gating. Simulation of a mixed POPC:cholesterol bilayer suggests that the hemichannel enhances the transbilayer mobility of vicinal cholesterols, increasing the likelihood of site-hopping and interleaflet flip-flop transitions.