Do small headgroups of phosphatidylethanolamine and phosphatidic acid lead to a similar folding pattern of the K(+) channel?

Phospholipid headgroups act as major determinants in proper folding of oligomeric membrane proteins. The K⁺-channel KcsA is the most popular model protein among these complexes. The presence of zwitterionic nonbilayer lipid phosphatidylethanolamine (PE) is crucial for efficient tetramerization and stabilization of KcsA in a lipid bilayer. In this study, the influence of PE on KcsA folding properties was analyzed by tryptophan fluorescence and acrylamide quenching experiments and compared with the effect of anionic phosphatidic acid (PA). The preliminary studies suggest that the small size and hydrogen bonding capability of the PE headgroup influences KcsA folding via a mechanism quite similar to that observed for anionic PA.     

Effects of Lipid Structure on the State of Aggregation of Potassium Channel KcsA

The state of aggregation of potassium channel KcsA was determined as a function of lipid:protein molar ratio in bilayer membranes of the zwitterionic lipid phosphatidylcholine (PC) and of the anionic lipid phosphatidylglycerol (PG). EPR (electron paramagnetic resonance) with spin-labeled phospholipids was used to determine the number of motionally restricted lipids per KcsA tetramer. Unexpectedly, this number decreased with a decreasing lipid:KcsA tetramer molar ratio in the range of 88:1 to 30:1, consistent with sharing of annular lipid shells and KcsA-KcsA contact at high mole fractions of protein. Fluorescence quenching experiments with brominated phospholipids showed a decrease in fluorescence quenching at low lipid:KcsA tetramer mole ratios, also consistent with KcsA-KcsA contact at high mole fractions of protein. The effects of low mole ratios of lipid seen in EPR and fluorescence quenching experiments were more marked in bilayers of PC than in bilayers of PG, suggesting stronger association of PG than PC with KcsA. This was confirmed by direct measurement of lipid association constants using spin-labeled phospholipids, showing higher association constants for all anionic lipids than for PC. The results show that the probability of contacts between KcsA tetramers will be very low at lipid:protein molar ratios that are typical of native biological membranes.     

Stability of KcsA tetramer depends on membrane lateral pressure

The potassium channel KcsA forms an extremely stable tetramer. Despite this high stability, it has been shown that the membrane-mimicking solvent 2,2,2-trifluoroethanol (TFE) can induce tetramer dissociation [Valiyaveetil, F. I., et al. (2002) Biochemistry 41, 10771-7, and Demmers, J. A. A., et al. (2003) FEBS Lett. 541, 69-77]. Here we have studied the effect of TFE on the structure and oligomeric state of the KcsA tetramer, reconstituted in different lipid systems. It was found that TFE changes the secondary and tertiary structure of KcsA and that it can dissociate the KcsA tetramer in all systems used. The tetramer is stabilized by a lipid bilayer as compared to detergent micelles. The extent of stabilization was found to depend on the nature of the lipids: a strong stabilizing effect of the nonbilayer lipid phosphatidylethanolamine (PE) was observed, but no effect of the charged phoshosphatidylglycerol (PG) as compared to phosphatidylcholine (PC) was found. To understand how lipids stabilize KcsA against TFE-induced tetramer dissociation, we also studied the effect of TFE on the bilayer organization in the various lipid systems, using (31)P and (2)H NMR. The observed lipid dependency was similar as was found for tetramer stabilization: PE increased the bilayer stability as compared to PC, while PG behaved similar to PC. Furthermore, it was found that TFE has a large effect on the acyl chain ordering. The results indicate that TFE inserts primarily in the membrane interface. We suggest that the lipid bilayer stabilizes the KcsA tetramer by the lateral pressure in the acyl chain region and that this stabilizing effect increases when a nonbilayer lipid like PE is present.     

Special Interaction of Anionic Phosphatidic Acid Promotes High Secondary Structure in Tetrameric Potassium Channel

Anionic phosphatidic acid (PA) has been shown to stabilize and bind stronger than phosphatidylglycerol via electrostatic and hydrogen bond interaction with the positively charged residues of potassium channel KcsA. However, the effects of these lipids on KcsA folding or secondary structure are not clear. In this study, the secondary structure analyses of KcsA potassium channel was carried out using circular dichroism spectroscopy. It was found that PA interaction leads to increases in α-helical and β-sheet content of KcsA protein. In PA, KcsA α-helical structure was further stabilized by classical membrane-active cosolvent trifluoroethanol followed by reduction in the β-sheet content indicating cooperative transformation from the β-sheet to an α-helical structure. The data further uncover the role of anionic PA in KcsA folding and provide mechanism by which strong hydrogen bonds/electrostatic interaction among PA headgroup and basic residues on lipid binding domains may induce high helical structure thereby altering the protein folding and increasing the stability of tetrameric assembly.     

The Role of Phosphatidic Acid and Cardiolipin in Stability of the Tetrameric Assembly of Potassium Channel KcsA

In this study, the roles of two anionic phospholipids—phosphatidic acid (PA), which is an important signaling molecule, and cardiolipin (CL), which plays a crucial role in the bioenergetics of the cell—in stabilizing the oligomeric structure of potassium channel KcsA were determined. The stability of KcsA was drastically increased as a function of PA or CL content (mol%) in phosphatidylcholine (PC) bilayers. Deletion of the membrane-associated N terminus significantly reduced channel stability at high levels of PA content; however, the intrinsic stability of this protein was marginally affected in the presence of CL. These studies indicate that the electrostatic-hydrogen bond switch between PA and N terminus, involving basic residues, is much stronger than the stabilizing effect of CL. Furthermore, the unique properties of the PA headgroup alter protein assembly and folding properties differently from the CL headgroup, and both lipids stabilize the tetrameric assembly via their specific interaction on the extra- or the intracellular side of KcsA.     

Monitoring the effects of strong cosolvent hexafluoroisopropanol in investigation of the tetrameric structure and stability of K-channel KcsA

Adsorption of small chain alcohols into lipid membranes significantly changes the conformational states of intrinsic membrane proteins. In this study, the effects of membrane-active strong cosolvent hexafluoroisopropanol (HFIP) on the intrinsic tetrameric stability of potassium channel KcsA were investigated. Presence of acidic phosphatidylglycerol (PG) in non-bilayer phosphatidylethanolamine (PE) or bilayer phosphatidylcholine (PC) significantly increased the tetrameric stability compared to zwitterionic pure PC bilayers. The stabilizing effect of PG in both lipid bilayers was completely abolished upon deletion of the membrane-anchored N-terminus. Tryptophan fluorescence and circular dichroism experiments indicated that HFIP destabilizes the tetramer possibly via drastic changes in the lateral pressure profile close to the membrane-water interface. The data suggest that HFIP disturbs the ionic, H-bonding and hydrophobic interactions among KcsA subunits where N-terminus presumably plays a crucial role in determining the channel proper folding and tetrameric structure via ionic/H-bond interactions between the helix dipole and the membrane lipids.     

The Role of Extramembranous Cytoplasmic Termini in Assembly and Stability of the Tetrameric K+-Channel KcsA

Membrane-active alcohol 2,2,2-trifluoroethanol has been proven to be an attractive tool in the investigation of the intrinsic stability of integral membrane protein complexes by taking K⁺-channel KcsA as a suitable and representative ion channel. In the present study, the roles of both cytoplasmic N and C termini in channel assembly and stability of KcsA were determined. The N terminus (1–18 residues) slightly increased tetramer stability via electrostatic interactions in the presence of 30 mol.% acidic phosphatidylglycerol (PG) in phosphatidylcholine lipid bilayer. Furthermore, the N terminus was found to be potentially required for efficient channel (re)assembly. In contrast, truncation of the C terminus (125–160 residues) greatly facilitated channel reversibility from either a partially or a completely unfolded state, and this domain was substantially involved in stabilizing the tetramer in either the presence or absence of PG in lipid bilayer. These studies provide new insights into how extramembranous parts play their crucial roles in the assembly and stability of integral membrane protein complexes.     

Anionic phospholipid interactions with the potassium channel KcsA: simulation studies

Molecular dynamics (MD) simulations have been used to unmask details of specific interactions of anionic phospholipids with intersubunit binding sites on the surface of the bacterial potassium channel KcsA. Crystallographic data on a diacyl glycerol fragment at this site were used to model phosphatidylethanolamine (PE), or phosphatidylglycerol (PG), or phosphatidic acid (PA) at the intersubunit binding sites. Each of these models of a KcsA-lipid complex was embedded in phosphatidyl choline bilayer and explored in a 20 ns MD simulation. H-bond analysis revealed that in terms of lipid-protein interactions PA > PG > PE and revealed how anionic lipids (PG and PA) bind to a site provided by two key arginine residues (R(64) and R(89)) at the interface between adjacent subunits. A 27 ns simulation was performed in which KcsA (without any lipids initially modeled at the R(64)/R(89) sites) was embedded in a PE/PG bilayer. There was a progressive specific increase over the course of the simulation in the number of H-bonds of PG with KcsA. Furthermore, two specific PG binding events at R(64)/R(89) sites were observed. The phosphate oxygen atoms of bound PG formed H-bonds to the guanidinium group of R(89), whereas the terminal glycerol H-bonded to R(64). Overall, this study suggests that simulations can help identify and characterize sites for specific lipid interactions on a membrane protein surface.     

Molecular determinants of tetramerization in the KcsA cytoplasmic domain

The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels.     

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA

All K⁺-channels are stabilized by K⁺-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K⁺-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K⁺-channel.     

Protein self-assembly and lipid binding in the folding of the potassium channel KcsA

Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl beta-d-maltoside (DDM) micelles [Barrera et al. (2005) Biochemistry 44, 14344-52]. Here we report that the transition from the folded tetramer to the unfolded monomer becomes completely reversible when KcsA is solubilized in mixed micelles composed of the detergent DDM and the lipids DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]). This result suggests that lipids may act as effectors in the tetramerization of KcsA. The observed reversibility allowed the determination of the standard free energy of the folding reaction of KcsA: DeltaG = 30.5 +/- 3.1 kcal x mol-1. We also observed that, prior to the unfolding of the tetramer, the presence of lower TFE concentrations causes the disassembly of supramolecular clusters of KcsA into the individual tetrameric molecules. Within the limits of experimental resolution, this is also a reversible process, but unlike the tetramer to monomer transition from above, the level of clustering is not influenced by the presence of solubilized lipids. These observations suggest a distinct role of the lipids in the different in vitro assembly steps (folding/tetramerization and clustering) of KcsA.     

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites.Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.     

Lipid–protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K⁺ channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid–protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~ 60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~ 80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (< 20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.     

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.     

Lipids in the structure,folding, and function of the KcsA K+ channel

Lipid molecules surround an ion channel in its native environment of cellular membranes. The importance of the lipid bilayer and the role of lipid protein interactions in ion channel structure and function are not well understood. Here we demonstrate that the bacterial potassium channel KcsA binds a negatively charged lipid molecule. We have defined the potential binding site of the lipid molecule on KcsA by X-ray crystallographic analysis of a complex of KcsA with a monoclonal antibody Fab fragment. We also demonstrate that lipids are required for the in vitro refolding of the KcsA tetramer from the unfolded monomeric state. The correct refolding of the KcsA tetramer requires lipids, but it is not dependent on negatively charged lipids as refolding takes place in the absence of such lipids. We confirm that the presence of negatively charged lipids is required for ion conduction through the KcsA potassium channel, suggesting that the lipid bound to KcsA is important for ion channel function.     

Small alcohols destabilize the KcsA tetramer via their effect on the membrane lateral pressure

Previously, it was shown that the tetrameric potassium channel KcsA when present in a lipid bilayer can be dissociated by trifluoroethanol [van den Brink-van der Laan, E., et al. (2004) Biochemistry 43, 4240-4250]. Here, we demonstrate that this is a general property of small alcohols. We found that small alcohols dissociate the KcsA tetramer, at a concentration that depends on their membrane affinity. Importantly, the efficiency of the alcohol-induced tetramer dissociation was found to correlate with the efficiency of both alcohol-induced bilayer leakage and acyl chain disordering. Our data suggest that the ability of small alcohols to induce KcsA tetramer dissociation and to function as anesthetics depends on their effect on the membrane lateral pressure.     

Phosphatidylglycerol lipids enhance folding of an alpha helical membrane protein

Membrane lipids are increasingly being recognised as active participants in biological events. The precise roles that individual lipids or global properties of the lipid bilayer play in the folding of membrane proteins remain to be elucidated, Here, we find a significant effect of phosphatidylglycerol (PG) on the folding of a trimeric α helical membrane protein from Escherichia coli diacylglycerol kinase. Both the rate and the yield of folding are increased by increasing the amount of PG in lipid vesicles. Moreover, there is a direct correlation between the increase in yield and the increase in rate; thus, folding becomes more efficient in terms of speed and productivity. This effect of PG seems to be a specific requirement for this lipid, rather than a charge effect. We also find an effect of single-chain lyso lipids in decreasing the rate and yield of folding. We compare this to our previous work in which lyso lipids increased the rate and yield of another membrane protein, bacteriorhodopsin. The contrasting effect of lyso lipids on the two proteins can be explained by the different folding reaction mechanisms and key folding steps involved. Our findings provide information on the lipid determinants of membrane protein folding.     

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.