Relation between flexibility and intrinsically disorder regions in thermosensitive TRP channels reveal allosteric effects.

How a protein propagates the conformational changes throughout its structure remains largely unknown. In thermosensitive TRP channels, this allosteric communication is triggered by ligand interaction or in response to temperature changes. Because dynamic allostery suggests a dynamic role of disordered regions, in this work we set out to thoroughly evaluate these regions in six thermosensitive TRP channels. Thus, by contrasting the intrinsic flexibility of the transmembrane region as a function of the degree of disorder in those proteins, we discovered several residues that do not show a direct correlation in both parameters. This kind of structural discrepancy revealed residues that are either reported to be dynamic, functionally relevant or are involved in signal propagation and probably part of allosteric networks. These discrepant, potentially dynamic regions are not exclusive of TRP channels, as this same correlation was found in the Kv Shaker channel.

Exploring Flexibility and Folding Patterns Throughout Time in Voltage Sensors.

The voltage-sensing domain (VSD) is a module capable of responding to changes in the membrane potential through conformational changes and facilitating electromechanical coupling to open a pore gate, activate proton permeation pathways, or promote enzymatic activity in some membrane-anchored phosphatases. To carry out these functions, this module acts cooperatively through conformational changes. The VSD is formed by four transmembrane segments (S1-S4) but the S4 segment is critical since it carries positively charged residues, mainly Arg or Lys, which require an aqueous environment for its proper function. The discovery of this module in voltage-gated ion channels (VGICs), proton channels (Hv1), and voltage sensor-containing phosphatases (VSPs) has expanded our understanding of the principle of modularity in the voltage-sensing mechanism of these proteins. Here, by sequence comparison and the evaluation of the relationship between sequence composition, intrinsic flexibility, and structural analysis in 14 selected representatives of these three major protein groups, we report five interesting differences in the folding patterns of the VSD both in prokaryotes and eukaryotes. Our main findings indicate that this module is highly conserved throughout the evolutionary scale, however: (1) segments S1 to S3 in eukaryotes are significantly more hydrophobic than those present in prokaryotes; (2) the S4 segment has retained its hydrophilic character; (3) in eukaryotes the extramembranous linkers are significantly larger and more flexible in comparison with those present in prokaryotes; (4) the sensors present in the kHv1 proton channel and the ciVSP phosphatase, both of eukaryotic origin, exhibit relationships of flexibility and folding patterns very close to the typical ones found in prokaryotic voltage sensors; and (5) archaeal channels KvAP and MVP have flexibility profiles which are clearly contrasting in the S3-S4 region, which could explain their divergent activation mechanisms. Finally, to elucidate the obscure origins of this module, we show further evidence for a possible connection between voltage sensors and TolQ proteins.

Non-canonical helical transitions and conformational switching are associated with characteristic flexibility and disorder indices in TRP and Kv channels.

Structural evidence and much experimental data have demonstrated the presence of non-canonical helical substructures (π and 310) in regions of great functional relevance both in TRP as in Kv channels. Through an exhaustive compositional analysis of the sequences underlying these substructures, we find that each of them is associated with characteristic local flexibility profiles, which in turn are implicated in significant conformational rearrangements and interactions with specific ligands. We found that α-to-π helical transitions are associated with patterns of local rigidity whereas α-to-310 transitions are mainly leagued with high local flexibility profiles. We also study the relationship between flexibility and protein disorder in the transmembrane domain of these proteins. By contrasting these two parameters, we located regions showing a sort of structural discrepancy between these similar but not identical protein attributes. Notably, these regions are presumably implicated in important conformational rearrangements during the gating in those channels. In that sense, finding these regions where flexibility and disorder are not proportional allows us to detect regions with potential functional dynamism. From this point of view, we highlighted some conformational rearrangements that occur during ligand binding events, the compaction, and refolding of the outer pore loops in several TRP channels, as well as the well-known S4 motion in Kv channels.

Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

Model lipid systems and their use to evaluate the phase state of biomembranes, their mechanical properties and the effect of non-conventional antibiotics: the case of daptomycin.

The lipid bilayer is the basis of the structure and function of the cell membrane. The study of the molecular phenomena that affect biological membranes has a great impact on the understanding of cellular physiology. To understand these phenomena, it has become increasingly necessary to develop simple synthetic models that allow the most basic details of such processes to be reproduced. In this short communication, we took advantage of the properties of two well-established lipid model systems, GUVs and SLBs, with compositions mimicking the cell membrane present in mammals and bacteria, to study the thermotropic phase behavior of lipids as well as the effect of daptomycin, a cyclic lipopeptide used as an antibiotic. The study of mechanical and thermodynamical properties of these model systems could contribute to establish a theoretical framework to develop more efficient strategies for biological control.

Voltage vs. Ligand I: Structural basis of the intrinsic flexibility of S3 segment and its significance in ion channel activation.

We systematically predict the internal flexibility of the S3 segment, one of the most mobile elements in the voltage-sensor domain. By analyzing the primary amino acid sequences of V-sensor containing proteins, including Hv1, TPC channels and the voltage-sensing phosphatases, we established correlations between the local flexibility and modes of activation for different members of the VGIC superfamily. Taking advantage of the structural information available, we also assessed structural aspects to understand the role played by the flexibility of S3 during the gating of the pore. We found that S3 flexibility is mainly determined by two specific regions: (1) a short NxxD motif in the N-half portion of the helix (S3a), and (2) a short sequence at the beginning of the so-called paddle motif where the segment has a kink that, in some cases, divide S3 into two distinct helices (S3a and S3b). A good correlation between the flexibility of S3 and the reported sensitivity to temperature and mechanical stretch was found. Thus, if the channel exhibits high sensitivity to heat or membrane stretch, local S3 flexibility is low. On the other hand, high flexibility of S3 is preferentially associated to channels showing poor heat and mechanical sensitivities. In contrast, we did not find any apparent correlation between S3 flexibility and voltage or ligand dependence. Overall, our results provide valuable insights into the dynamics of channel-gating and its modulation.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels.

In the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins. A highly flexible S4-like segment exhibiting Lys instead Arg for these membrane proteins is reported. Sequence analysis indicates that, in addition to this S4-like segment, TolQ proteins also show similarity with specific motifs in S2 and S3 from typical V-sensors. Notably, S3 flexibility profiles from typical VSDs and S3-like in TolQ proteins are also similar. Interestingly, TolQ from early divergent prokaryotes are comparatively more flexible than those in modern counterparts or true V-sensors. Regarding the PD, we also found that 2TM K+-channels in early prokaryotes are considerably more flexible than the ones in modern microbes, and such flexibility is comparable to the one present in CNG channels. Voltage dependence is mainly exhibited in prokaryotic CNG channels whose VSD is rigid whereas the eukaryotic CNG channels are considerably more flexible and poorly V-dependent. The implication of the flexibility present in CNG channels, their sensitivity to cyclic nucleotides and the cation selectivity are discussed. Finally, we generated a structural model of the putative cyclic nucleotide-modulated ion channel, which we coined here as AqK, from the thermophilic bacteria Aquifex aeolicus, one of the earliest diverging prokaryotes known. Overall, our analysis suggests that V-sensors in CNG-like channels were essentially rigid in early prokaryotes but raises the possibility that this module was probably part of a very flexible stator protein of the bacterial flagellum motor complex.

Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides.

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

Complex Phase Behavior of GUVs Containing Different Sphingomyelins.

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

Side chain flexibility and coupling between the S4-S5 linker and the TRP domain in thermo-sensitive TRP channels: Insights from protein modeling.

The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side-chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4-S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly-conserved Trp during this coupling. Our analysis suggests an important role of the S4-S5L flexibility in the thermosensitivity, where heat-activated channels possess rigid S4-S5 linkers, whereas cold-activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4-S5L, importantly the central Pro. Proteins 2017; 85:630-646. © 2016 Wiley Periodicals, Inc.

K⁺-dependent selectivity and external Ca²âº block of Shab K⁺ channels.

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na(o)/K(i) or Na(o)/Rb(i), conditions. However, when internal K⁺ is replaced by Cs⁺ (Na(o)/Cs(i)), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²âº ions, and compare the effect that internal K⁺ replacement exerts on both Ca²âº and TEA block. Our observations indicate that Ca²âº blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²âº is probably coordinated by main chain carbonyls of the pore's first K⁺-binding site.

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study.

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study.

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

Conservation analysis of residues in the S4-S5 linker and the terminal part of the S5-P-S6 pore modulus in Kv and HCN channels: flexible determinants for the electromechanical coupling.

Protein mobility is important to achieve protein function. Intrinsic flexibility associated with motion underlies this important issue and the analysis of side chain flexibility gives insights to understand it. In this work, the S5-P-S6 pore modulus (PM) of members of Kv and HCN channels was examined by a combination of sequence alignment, residue composition analysis, and intrinsic side chain flexibility. The PM sequences were organized as a database that was used to reveal and correlate the functional diversity of each analyzed family. Specifically, we focused our attention on the crucial role of the S4-S5 linker and its well-described interaction with the S6 T during the electromechanical coupling. Our analysis suggests the presence of a Gly-hinge in the middle of the S4-S5 linkers. This apparent Gly-hinge links a flexible N-terminal segment with a rigid C-terminal one, although in Kv7 channels, the latter segment is even more flexible. Instead, HCN channels exhibit a putative Thr-hinge and is rich in aromatic residues, in consequence, their linker is more rigid. Concerning S6, we confirm the presence of the two flexible kinks previously described and we provide the complete segmental flexibility profiles for the different families. Our results are discussed in terms of the relation between residue composition, conservation, and local conformational flexibility. This provides important insights to understand and differentiate the characteristic gating properties of these channels as well as their implications in cell physiology.

Ether- versus ester-linked phospholipid bilayers containing either linear or branched apolar chains.

We studied the properties of bilayers formed by ether-and ester-containing phospholipids, whose hydrocarbon chains can be either linear or branched, using sn-1,2 dipalmitoyl, dihexadecyl, diphytanoyl, and diphytanyl phosphatidylcholines (DPPC, DHPC, DPhoPC, and DPhPC, respectively) either pure or in binary mixtures. Differential scanning calorimetry and confocal fluorescence microscopy of giant unilamellar vesicles concurred in showing that equimolar mixtures of linear and branched lipids gave rise to gel/fluid phase coexistence at room temperature. Mixtures containing DHPC evolved in time (0.5 h) from initial reticulated domains to extended solid ones when an equilibrium was achieved. The nanomechanical properties of supported planar bilayers formed by each of the four lipids studied by atomic force microscopy revealed average breakdown forces Fb decreasing in the order DHPC ≥ DPPC > DPhoPC >> DPhPC. Moreover, except for DPPC, two different Fb values were found for each lipid. Atomic force microscopy imaging of DHPC was peculiar in showing two coexisting phases of different heights, probably corresponding to an interdigitated gel phase that gradually transformed, over a period of 0.5 h, into a regular tilted gel phase. Permeability to nonelectrolytes showed that linear-chain phospholipids allowed a higher rate of solute + water diffusion than branched-chain phospholipids, yet the former supported a smaller extent of swelling of the corresponding vesicles. Ether or ester bonds appeared to have only a minor effect on permeability.

Mechanical properties of lipid bilayers and regulation of mechanosensitive function: from biological to biomimetic channels.

Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.

Toward understanding protocell mechanosensation.

Mechanosensitive (MS) channels can prevent bacterial bursting during hypo-osmotic shocks by responding to increases in lateral tension at the membrane level through an integrated and coordinated opening mechanism. Mechanical regulation in protocells could have been one of the first mechanisms to evolve in order to preserve their integrity against changing environmental conditions. How has the rich functional diversity found in present cells been created throughout evolution, and what did the primordial MS channels look like? This review has been written with the aim of identifying which factors may have been important for the appearance of the first osmotic valve in a prebiotic context, and what this valve may have been like. It highlights the mechanical properties of lipid bilayers, the association of peptides as aggregates in membranes, and the conservation of sequence motifs as central aspects to understand the evolution of proteins that gate below the tension required for spontaneous pore formation and membrane rupture. The arguments developed here apply to both MscL and MscS homologs, but could be valid to mechano-susceptible proteins in general.

Induction of a fast inactivation gating on delayed rectifier Shab K(+) channels by the anti-inflammatory drug celecoxib.

Celecoxib is a drug designed to selectively inhibit COX-2, an inflammation-inducible cyclooxygenase isoform, over the constitutively expressed COX-1 isoform. In addition to this selective inhibition it is now known that celecoxib exerts a variety of effects on several types of ion channels, thus producing secondary physiological effects. In this work we demonstrate that at therapeutically relevant concentrations celecoxib interacts with Shab K(+) channels specifically promoting a fast inactivation gating (without blocking the pore or significantly affecting other gating processes). At least two celecoxib molecules bind to each channel promoting a fast inactivation that develops from both open and closed states. Channel inactivation in turn causes a reduction of the size of I(K). Taken together, our observations show that in addition to its intended therapeutic target celecoxib is a useful tool to further study the mechanism of Shab channel inactivation.