Membrane-inserted State Research Articles

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis.

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

Not sorcery after all: Roles of multiple charged residues in membrane insertion of gasdermin-A3.

Gasdermins execute programmatory cell death, known as pyroptosis, by forming medium-sized membrane pores. Recently, the molecular structure of those pores as well as the diversity in their shape and size have been revealed by cryoTEM and atomic force microscopy, respectively. Even though a growth of smaller to larger oligomers and reshaping from slits to rings could be documented, the initiation of the gasdermin pore formation remains a mystery. In one hypothesis, gasdermin monomers insert into membranes before associating into oligomeric pores. In the other hypothesis, gasdermin oligomers preassemble on the membrane surface prior to membrane insertion. Here, by studying the behavior of monomeric membrane-inserted gasdermin-A3 (GSDMA3), we unveil that a monomeric gasdermin prefers the membrane-adsorbed over the membrane-inserted state. Our results thus support the hypothesis of oligomers preassembling on the membrane surface before membrane penetration. At the same time, our simulations of small membrane-inserted arcs of GSDMA3 suggest that the inserting oligomer can be small and does not have to comprise a full ring of approximately 26–30 subunits. Moreover, our simulations have revealed an astonishingly large impact of salt-bridge formation and protein surroundings on the transmembrane passage of charged residues, reducing the energetic cost by up to 53% as compared to their free forms. The here observed free energy barrier of mere 5.6 kcal/mol for the membrane insertion of monomeric GSDMA3 explains the surprising ability of gasdermins to spontaneously self-insert into cellular membranes.

Molecular Dynamics Study of Lipid and Cholesterol Reorganization Due to Membrane Binding and Pore Formation by Listeriolysin O.

Pore-forming toxins are proteinsexpressed by bacteria to primarily cause infections in the host cell. Cholesterol-dependent cytolysins (CDCs) are a class of proteins whose pore-forming ability requires the presence of cholesterol in the membrane. Upon binding to the target cell, cholesterol-recognizing residues in the membrane binding D4 subdomain assist in stabilizing both the pre-pore and pore states which occur during protein oligomerization on the cell membrane. Super resolution-stimulated emission depletion (STED) microscopy experiments (Sarangi et al. in Langmuir, 32:9649-9657, 2016) on supported lipid bilayers have shown that listeriolysin (LLO), a CDC expressed by Listeria monocytogenes, a food-borne pathogen, induces both spatial and dynamic heterogeneity in bilayer membranes. Here, we use all-atom molecular dynamics simulations to explore molecular details of the induced membrane reorganization by considering two distinct states of the oligomerized LLO protein in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): cholesterol membrane. In the membrane bound (MB) state, four D4 subunits are placed at the bilayer interface in a pre-pore configuration and the membrane-inserted (MI) state consists of a tetrameric arc-like pore configuration. By analyzing lipid-order parameters, mobilities, and diffusion coefficients, we examine the induced spatial heterogeneity that occurs in both the MB and MI states. This heterogeneity is primarily driven by the local density enhancement of cholesterol in the vicinity of the MB, D4 subunits leading to distinct differences in lipid and cholesterol mobility across the two leaflets as well as enhanced lipid mobilities in regions where cholesterol is depleted. The leaflet-induced heterogeneity is greater for the MB state when compared with the MI state and the dynamic variations are more pronounced in the extracellular leaflet when compared with the cytosolic leaflet. Our study provides molecular-level insights into the inhomogeneity and perturbation induced in bilayer membranes upon LLO binding and pore formation and is expected to represent trends across PFTs in the broad CDC subclass of proteins.

Coarse-grained MD simulations reveal beta-amyloid fibrils of various sizes bind to interfacial liquid-ordered and liquid-disordered regions in phase separated lipid rafts with diverse membrane-bound conformational states

The membrane binding behaviors of beta-amyloid fibrils, dimers to pentamers, from solution to lipid raft surfaces, were investigated using coarse-grained (CG) MD simulations. Our CG rafts contain phospholipid, cholesterol (with or without tail- or headgroup modifications), and with or without asymmetrically distributed monosialotetrahexosylganglioside (GM1). All rafts exhibited liquid-ordered (Lo), liquid-disordered (Ld), and interfacial Lo/Ld (Lod) domains, with domain sizes depending on cholesterol structure. For rafts without GM1, all fibrils bound to the Lod domains. Specifically, dimer fibrils bound exclusively via the C-terminal, while larger fibrils could bind via other protein regions. Interestingly, a membrane-inserted state was detected for a trimer fibril in a raft with tail-group modified cholesterol. For rafts containing GM1, fibrils bound either to the GM1-clusters, with numerous membrane-bound conformations, or to the non-GM1-containing-Lod domains via the C-terminal. Our results indicate beta-amyloid fibrils bind to Lod domains or GM1, with diversified membrane-bound conformations, in structurally heterogeneous lipid membranes.

Membrane-Induced p Ka Shifts in wt-pHLIP and Its L16H Variant.

The pH (low) insertion peptides (pHLIPs) is a family of peptides that are able to insert into a lipid bilayer at acidic pH. The molecular mechanism of pHLIPs insertion, folding, and stability in the membrane at low pH is based on multiple protonation events, which are challenging to study at the molecular level. More specifically, the relation between the experimental p K of insertion (p Kexp) of pHLIPs and the p Ka of the key residues is yet to be clarified. We carried out a computational study, complemented with new experimental data, and established the influence of (de)protonation of titrable residues on the stability of the peptide membrane-inserted state. Constant-pH molecular dynamics simulations were employed to calculate the p Ka values of these residues along the membrane normal. In the wt-pHLIP, we identified Asp14 as the key residue for the stability of the membrane-inserted state, and its p Ka value is strongly correlated with the experimental p Kexp measured in thermodynamics studies. Also, in order to narrow down the pH range at which pHLIP is stable in the membrane, we designed a new pHLIP variant, L16H, where Leu in the 16th position was replaced by a titrable His residue. Our results showed that the L16H variant undergoes two transitions. The calculated p Ka and experimentally observed p Kexp values are in good agreement. Two distinct p Kexp values delimit a pH range where the L16H peptide is stably inserted in the membrane, while, outside this range, the membrane-inserted state is destabilized and the peptide exits from the bilayer. pHLIP peptides have been successfully used to target cancer cells for the delivery of diagnostics and therapeutic agents to acidic tumors. The fine-tuning of the stability of the pHLIP inserted state and its restriction to a narrow well-defined pH range might allow the design of new peptides, able to discriminate between tissues with different extracellular pH values.

The SecA protein deeply penetrates into the SecYEG channel during insertion, contacting most channel transmembrane helices and periplasmic regions

The bacterial Sec-dependent system is the major protein-biogenic pathway for protein secretion across the cytoplasmic membrane or insertion of integral membrane proteins into the phospholipid bilayer. The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial with conflicting claims from biochemical and structural studies regarding the depth and extent of SecA insertion into SecYEG during ongoing protein transport. Here we utilized site-specific in vivo photo-crosslinking to thoroughly map SecY regions that are in contact with SecA during its insertion cycle. An arabinose-inducible, rapidly folding OmpA-GFP chimera was utilized to jam the SecYEG channels with an arrested substrate protein to "freeze" them in their SecA-inserted state. Examination of 117 sites distributed throughout SecY indicated that SecA not only interacts extensively with the cytosolic regions of SecY as shown previously, but it also interacts with most of the transmembrane helices and periplasmic regions of SecY, with a clustering of interaction sights around the lateral gate and pore ring regions. Our observations support previous reports of SecA membrane insertion during in vitro protein transport as well as those documenting the membrane penetration properties of this protein. They suggest that one or more SecA regions transiently integrate into the heart of the SecY channel complex to span the membrane to promote the protein transport cycle. These findings indicate that high-resolution structural information about the membrane-inserted state of SecA is still lacking and will be critical for elucidating the bacterial protein transport mechanism.

A hemi-fission intermediate links two mechanistically distinct stages of membrane fission.

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate1,2, characterized by a ‘stalk’ in which only the inner monolayers of the two compartments have merged to form a localized non-bilayer connection1-3. Formation of the hemi-fission intermediate requires energy input from proteins catalyzing membrane remodeling; however the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analyzed how the GTPase cycle of dynamin, the prototypical membrane fission catalyst4-6, is directly coupled to membrane remodeling. We used intra-molecular chemical cross-linking to stabilize dynamin in its GDP•AlF4--bound transition-state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fueled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction7-8, the force bimodality might constitute a general paradigm for leakage-free membrane remodeling.

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.

Bilayer surface association of the pHLIP peptide promotes extensive backbone desolvation and helically-constrained structures

Despite their presence in many aspects of biology, the study of membrane proteins lags behind that of their soluble counterparts. Improving structural analysis of membrane proteins is essential. Deep-UV resonance Raman (DUVRR) spectroscopy is an emerging technique in this area and has demonstrated sensitivity to subtle structural transitions and changes in protein environment. The pH low insertion peptide (pHLIP) has three distinct structural states: disordered in an aqueous environment, partially folded and associated with a lipid membrane, and inserted into a lipid bilayer as a transmembrane helix. While the soluble and membrane-inserted forms are well characterized, the partially folded membrane-associated state has not yet been clearly described. The amide I mode, known to be sensitive to protein environment, is the same in spectra of membrane-associated and membrane-inserted pHLIP, indicating comparable levels of backbone dehydration. The amide S mode, sensitive to helical structure, indicates less helical character in the membrane-associated form compared to the membrane-inserted state, consistent with previous findings. However, the structurally sensitive amide III region is very similar in both membrane-associated and membrane-inserted pHLIP, suggesting that the membrane-associated form has a large amount of ordered structure. Where before the membrane-associated state was thought to contain mostly unordered structure and reside in a predominantly aqueous environment, we have shown that it contains a significant amount of ordered structure and rests deeper within the lipid membrane.

Évaluation du contrôle de l’asthme en consultation de pneumologie au Cameroun

Despite their presence in many aspects of biology, the study of membrane proteins lags behind that of their soluble counterparts. Improving structural analysis of membrane proteins is essential. Deep-UV resonance Raman (DUVRR) spectroscopy is an emerging technique in this area and has demonstrated sensitivity to subtle structural transitions and changes in protein environment. The pH low insertion peptide (pHLIP) has three distinct structural states: disordered in an aqueous environment, partially folded and associated with a lipid membrane, and inserted into a lipid bilayer as a transmembrane helix. While the soluble and membrane-inserted forms are well characterized, the partially folded membrane-associated state has not yet been clearly described. The amide I mode, known to be sensitive to protein environment, is the same in spectra of membrane-associated and membrane-inserted pHLIP, indicating comparable levels of backbone dehydration. The amide S mode, sensitive to helical structure, indicates less helical character in the membrane-associated form compared to the membrane-inserted state, consistent with previous findings. However, the structurally sensitive amide III region is very similar in both membrane-associated and membrane-inserted pHLIP, suggesting that the membrane-associated form has a large amount of ordered structure. Where before the membrane-associated state was thought to contain mostly unordered structure and reside in a predominantly aqueous environment, we have shown that it contains a significant amount of ordered structure and rests deeper within the lipid membrane.

Comparison of Membrane Insertion Pathways of the Apoptotic Regulator Bcl-xL and the Diphtheria Toxin Translocation Domain

The diphtheria toxin translocation domain (T-domain) and the apoptotic repressor Bcl-xL are membrane proteins that adopt their final topology by switching folds from a water-soluble to a membrane-inserted state. While the exact molecular mechanisms of this transition are not clearly understood in either case, the similarity in the structures of soluble states of the T-domain and Bcl-xL led to the suggestion that their membrane insertion pathways will be similar, as well. Previously, we have applied an array of spectroscopic methods to characterize the pH-triggered refolding and membrane insertion of the diphtheria toxin T-domain. Here, we use the same set of methods to describe the membrane insertion pathway of Bcl-xL, which allows us to make a direct comparison between both systems with respect to the thermodynamic stability in solution, pH-dependent membrane association, and transmembrane insertion. Thermal denaturation measured by circular dichroism indicates that, unlike the T-domain, Bcl-xL does not undergo a pH-dependent destabilization of the structure. Förster resonance energy transfer measurements demonstrate that Bcl-xL undergoes reversible membrane association modulated by the presence of anionic lipids, suggesting that formation of the membrane-competent form occurs close to the membrane interface. Membrane insertion of the main hydrophobic helical hairpin of Bcl-xL, α5-α6, was studied by site-selective attachment of environment-sensitive dye NBD. In contrast to the insertion of the corresponding TH8-TH9 hairpin into the T-domain, insertion of α5-α6 was found not to depend strongly on the presence of anionic lipids. Taken together, our results indicate that while Bcl-xL and the T-domain share structural similarities, their modes of conformational switching and membrane insertion pathways are distinctly different.

Exclusively membrane-inserted state of an uncleavable Tat precursor protein suggests lateral transfer into the bilayer from the translocon

In bacteria, the export of proteins by the twin-arginine translocase (Tat) pathway is directed by cleavable N-terminal signal peptides. We studied the relationship between transport and maturation using a substrate, YedY, that contains an Ala > Leu substitution at the -1 position of the signal peptide. This blocks maturation and leads to the accumulation of a membrane-bound precursor form with the mature domain exposed to the periplasm. Its accumulation does not block transport of other Tat substrates, indicating that exit from the translocation channel has taken place, and the precursor protein is fir mLy integrated into the membrane bilayer. The membrane-integrated nature of the precursor, and complete absence of precursor protein in the periplasm, strongly suggest that the precursor has undergone lateral transfer into the bilayer during translocation. We propose that subsequent proteolytic processing releases the mature protein into the periplasm. A delay in processing results in an inhibition of cell growth, emphasizing a requirement for efficient maturation of Tat substrates.

Novel Concept of Delivery of Diagnostic and Therapeutic Agents to Cells in Acidic Diseased Tissue using Energy of Membrane-Associated Folding

We are developing a novel technology for selective delivery of imaging probes and membrane-impermeable molecules to cells with low extracellular pH. It is based on action of water-soluble membrane peptide, pHLIP® (pH [Low] Insertion Peptide), which inserts and folds in cellular membrane at slightly acidic environment, characteristic for various pathological states including cancer and ischemic myocardium. pHLIP possess dual delivery capability. Imaging agents (fluorescent, PET, SPECT or MRI) could be attached to the N-terminus of the peptide to mark tumor mass and tumor margins with high precision. At the same time, therapeutic molecules attached to the C-inserting end, could be moved across membrane to reach cytoplasmic target. Among translocated molecules are synthetic cyclic peptides, gene regulation agent (peptide nucleic acid) and phalla- and amanita toxins with hydrophobicity tuned by attachment of fatty acids for optimum delivery. We performed sequence variation and investigated 16 pHLIP variants with main goals of understanding the main principles of peptide-lipid interactions and tune delivery capability of pHLIP. The biophysical studies including thermodynamics and kinetics of the peptides interaction with a lipid bilayer of liposomes and cellular membranes were carried out. We found that peptides association to membrane at neutral and low pH could be modulated by 3-4 times. The apparent pK of transition from surface bound to membrane-inserted state could be tuned from 6.5 to 4.5. The rate of peptide's insertion across a bilayer could be enhanced 100 times compared to parent pHLIP. As a result, blood clearance and tumor targeting were modulated in a significant degree. The work is supported by NIH grants CA133890 to OAA, DME, YRK.

Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes.

Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Dissecting the Membrane Binding and Insertion Kinetics of a pHLIP Peptide

When it is bound to lipid bilayers, the conformation and location of the membrane pH (low) insertion peptide (pHLIP) depend on pH. This unique feature allows us to explicitly measure the kinetics leading to different membrane-bound states of pHLIP using a model membrane and stopped-flow technique. Our results show that the membrane association kinetics of pHLIP are multiexponential and are consistent with a parallel membrane interaction mechanism. Interestingly, our results also show that the overall rate at which the membrane-inserted state is formed is almost identical to that of formation of the surface-bound state, while prebinding slows the rate of peptide insertion.

Deregulation of the SecYEG Translocation Channel upon Removal of the Plug Domain

Previous studies have shown that the SecY plug is displaced from the center of the SecYEG channel during polypeptide translocation. The structural and functional consequences of the deletion of the plug are now examined. Both in vivo and in vitro observations indicate that the plug domain is not essential to the function of the translocon. In fact, deletion of the plug confers to the cell and to the membranes a Prl-like phenotype: reduced proton-motive force dependence of translocation, increased membrane insertion of SecA, diminished requirement for functional leader peptide, and weakened SecYEG subunit association. Although the plug domain does not seem essential, locking the plug in the center of the channel inactivates the translocon. Thus, the SecY plug is important to regulate the activity of the channel and to confer specificity to the translocation reaction. We propose that the plug contributes to the gating mechanism of the channel by maintaining the structure of the SecYEG complex in a compact closed state.

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5–10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.

Mapping Functional Domains of Chloride Intracellular Channel (CLIC) Proteins in Vivo

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A < 70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ‘‘PTM‘‘ domain, named after an amphipathic putative trans membrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.

The role of a conserved histidine–tyrosine interhelical interaction in the ion channel domain of δ‐endotoxins fromBacillus thuringiensis

The delta-endotoxin proteins are produced by Bacillus thuringiensis during the sporulation phase of its life cycle. These proteins exhibit insecticidal activity through receptor-mediated ion channel formation. The mode of action of these proteins requires the conversion of the protein from a water-soluble conformation to a membrane-inserted conformation. While there is X-ray structure information for the soluble protein, no detailed structure exists for the membrane-inserted protein. However, based on peptide studies, an umbrella model for the membrane-inserted state has been proposed. Here, we investigated the role of a conserved hydrogen bond interaction between two helices that are suggested to undergo a large conformational change upon membrane insertion. Mutation of either the histidine or the tyrosine resulted in a protein that has significantly reduced bioactivity, increased overall flexibility, and significantly reduced stability. These data highlight an important role for this interaction in the overall stability of the protein. Additionally, the conservation of histidine and tyrosine in these positions may suggest a functional role for the interaction in the conformational switching from soluble to membrane protein.