Helical Hairpin Research Articles

Structure–function analysis of tRNA t6A-catalysis, assembly and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

The universal N6-threonylcarbamoyladenosine (t6A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t6A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t6A biosynthesis using recombinant TsaC, TsaD–TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD2B2 tetramer. Our TsaD2B2–TsaE–tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1–α2 of TsaD. A. aeolicus TsaD2B2 or TsaDB possesses a basal divalent ion-dependent t6A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t6A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t6A-catalytic activity of TsaD2B2–tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

Exploration of Tertiary Structure in Sequence-Defined Polymers Using Molecular Dynamics Simulations.

Peptoids are a class of sequence-defined biomimetic polymers with peptide-like backbones and side chains located on backbone nitrogens rather than alpha carbons. These materials demonstrate a strong ability for precise control of single-chain structure, multiunit self-assembly, and macromolecular assembly through careful tuning of sequence due to the diversity of available side chains, although the driving forces behind these assemblies are often not understood. Prior experimental work has shown that linked 15mer peptoids can mimic the protein helical hairpin structure by leveraging the chirality-inducing nature of bulky side chains and hydrophobicity, but there are still gaps in our understanding of the relationship between sequence, stability, and particular secondary or tertiary structure. We present a molecular dynamics (MD) study on the folding behavior of these polymers into hairpins, discussing the differences in structure from sequences with various characteristics in water and acetonitrile, and then compare the handedness preference of common helical motifs between solvents.

Computational design and experimental confirmation of a disulfide-stapled YAP helixα1-trap derived from TEAD4 helical hairpin to selectively capture YAP α1-helix with potent antitumor activity.

Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.

Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.

Multiple variants of the type VII secretion system in Gram-positive bacteria.

Type VII secretion systems (T7SS) are found in bacteria across the Bacillota and Actinomycetota phyla and have been well described in Staphylococcus aureus, Bacillus subtilis, and pathogenic mycobacteria. The T7SS from Actinomycetota and Bacillota share two common components, a membrane-bound EccC/EssC ATPase and EsxA, a small helical hairpin protein of the WXG100 family. However, they also have additional phylum-specific components, and as a result they are termed the T7SSa (Actinomycetota) and T7SSb (Bacillota), respectively. Here, we identify additional organizations of the T7SS across these two phyla and describe eight additional T7SS subtypes, which we have named T7SSc-T7SSj. T7SSd is found exclusively in Actinomycetota including the Olselnella and Bifodobacterium genus, whereas the other seven are found only in Bacillota. All of the novel subtypes contain the canonical ATPase (TsxC) and the WXG100-family protein (TsxA). Most of them also contain a small ubiquitin-related protein, TsxB, related to the T7SSb EsaB/YukD component. Protein kinases, phosphatases, and forkhead-associated (FHA) proteins are often encoded in the novel T7SS gene clusters. Candidate substrates of these novel T7SS subtypes include LXG-domain and RHS proteins. Predicted substrates are frequently encoded alongside genes for additional small WXG100-related proteins that we speculate serve as cosecretion partners. Collectively our findings reveal unexpected diversity in the T7SS in Gram-positive bacteria.

SARS-CoV-2 Fusion Peptide Conjugated to a Tetravalent Dendrimer Selectively Inhibits Viral Infection.

Fusion is a key event for enveloped viruses, through which viral and cell membranes come into close contact. This event is mediated by viral fusion proteins, which are divided into three structural and functional classes. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein belongs to class I fusion proteins, characterized by a trimer of helical hairpins and an internal fusion peptide (FP), which is exposed once fusion occurs. Many efforts have been directed at finding antivirals capable of interfering with the fusion mechanism, mainly by designing peptides on the two heptad-repeat regions present in class I viral fusion proteins. Here, we aimed to evaluate the anti-SARS-CoV-2 activity of the FP sequence conjugated to a tetravalent dendrimer through a classical organic nucleophilic substitution reaction (SN2) using a synthetic bromoacetylated peptide mimicking the FP and a branched scaffold of poly-L-Lysine functionalized with cysteine residues. We found that the FP peptide conjugated to the dendrimer, unlike the monomeric FP sequence, has virucidal activity by impairing the attachment of SARS-CoV-2 to cells. Furthermore, we found that the peptide dendrimer does not have the same effects on other coronaviruses, demonstrating that it is selective against SARS-CoV-2.

Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the determination of the three-dimensional structure of AIP56 shows that, instead of a two-domain organization suggested by previous studies, AIP56 has three-domains: a non-LEE encoded effector C (NleC)-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only upon endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation.

Cryo‐EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram‐positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton‐coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo‐EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate‐free QacA, complexed to two single‐domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward‐open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.

WS04.02 Impact of cholesterol on the functioning of CFTR modulators

Objectives: Orkambi is a combination of two CFTR modulators: corrector VX-809 and potentiator VX-770. Both drugs have been separately shown to bind CFTR at a protein-lipid interface, albeit at various locations on the channel. The cholesterol-rich membrane surrounding CFTR plays an important role in its proper folding. However, minimal research has been done to understand the potential role cholesterol plays in facilitating drug-CFTR interactions. Here we investigated the role cholesterol plays in Orkambi’s interaction with mammalian membrane and in folding of helical hairpin constructs consisting of transmembrane segments 3 and 4 (TM3/4) of CFTR’s transmembrane domain 1.

Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins.

Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 hr at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane homodimer for 9 hr at 12 pN and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling analysis, respectively. The Kramers rate framework yields a considerably low-speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.

Two modes of fusogenic action for influenza virus fusion peptide.

The entry of influenza virus into the host cell requires fusion of its lipid envelope with the host membrane. It is catalysed by viral hemagglutinin protein, whose fragments called fusion peptides become inserted into the target bilayer and initiate its merging with the viral membrane. Isolated fusion peptides are already capable of inducing lipid mixing between liposomes. Years of studies indicate that upon membrane binding they form bend helical structure whose degree of opening fluctuates between tightly closed hairpin and an extended boomerang. The actual way in which they initiate fusion remains elusive. In this work we employ atomistic simulations of wild type and fusion inactive W14A mutant of influenza fusion peptides confined between two closely apposed lipid bilayers. We characterise peptide induced membrane perturbation and determine the potential of mean force for the formation of the first fusion intermediate, an interbilayer lipid bridge called stalk. Our results demonstrate two routes through which the peptides can lower free energy barrier towards fusion. The first one assumes peptides capability to adopt transmembrane configuration which subsequently promotes the creation of a stalk-hole complex. The second involves surface bound peptide configuration and proceeds owing to its ability to stabilise stalk by fitting into the region of extreme negative membrane curvature resulting from its formation. In both cases, the active peptide conformation corresponds to tight helical hairpin, whereas extended boomerang geometry appears to be unable to provide favourable thermodynamic effect. The latter observation offers plausible explanation for long known inactivity of boomerang-stabilising W14A mutation.

Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide-protein or protein-protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E.coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E.coli and mammalian cells.

Structural basis of ELKS/Rab6B interaction and its role in vesicle capturing enhanced by liquid-liquid phase separation

ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts with the vesicular traffic regulator, the Rab6 GTPase, the molecular basis governing ELKS-mediated trafficking of Rab6-coated vesicles, has remained unclear. In this study, we solved the Rab6B structure in complex with the Rab6-binding domain of ELKS1, revealing that a C-terminal segment of ELKS1 forms a helical hairpin to recognize Rab6B through a unique binding mode. We further showed that liquid-liquid phase separation (LLPS) of ELKS1 allows it to compete with other Rab6 effectors for binding to Rab6B and accumulate Rab6B-coated liposomes to the protein condensate formed by ELKS1. We also found that the ELKS1 condensate recruits Rab6B-coated vesicles to vesicle-releasing sites and promotes vesicle exocytosis. Together, our structural, biochemical, and cellular analyses suggest that ELKS1, via the LLPS-enhanced interaction with Rab6, captures Rab6-coated vesicles from the cargo transport machine for efficient vesicle release at exocytotic sites. These findings shed new light on the understanding of spatiotemporal regulation of vesicle trafficking through the interplay between membranous structures and membraneless condensates.

Synthesis and Crystallographic Characterization of Helical Hairpin Oligourea Foldamers.

Oligomers designed to form a helix-turn-helix super-secondary structure have been prepared by covalently bridging aliphatic oligourea foldamer helices with either rigid aromatic or more flexible aliphatic spacers. The relative helix orientation in these dimers has been investigated at high resolution using X-ray diffraction analysis. In several cases, racemic crystallography was used to facilitate crystallization and structure determination. All structures were solved by direct methods. Well-defined parallel helical hairpin motifs were observed in all cases when 4,4'-methylene diphenyl diisocyanate was employed as a dimerizing agent, irrespective of primary sequence and chain length.

Conformation and Orientation of Antimicrobial Peptides MSI-594 and MSI-594A in a Lipid Membrane.

There is significant interest in the development of antimicrobial compounds to overcome the increasing bacterial resistance to conventional antibiotics. Studies have shown that naturally occurring and de novo-designed antimicrobial peptides could be promising candidates. MSI-594 is a synthetic linear, cationic peptide that has been reported to exhibit a broad spectrum of antimicrobial activities. Investigation into how MSI-594 disrupts the cell membrane is important for better understanding the details of this antimicrobial peptide (AMP)'s action against bacterial cells. In this study, we used two different synthetic lipid bilayers: zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 7:3 POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (POPG). Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) were used to determine the orientations of MSI-594 and its analogue MSI-594A associated with zwitterionic POPC and anionic 7:3 POPC/POPG lipid bilayers. The simulated ATR-FTIR and SFG spectra using nuclear magnetic resonance (NMR)-determined structures were compared with experimental spectra to optimize the bent angle between the N- (1-11) and C- (12-24) termini helices and the membrane orientations of the helices; since the NMR structure of the peptide was determined from lipopolysaccharide (LPS) micelles, the optimization was needed to find the most suitable conformation and orientation in lipid bilayers. The reported experimental results indicate that the optimized MSI-594 helical hairpin structure adopts a complete lipid bilayer surface-bound orientation (denoted "face-on") in both POPC and 7:3 POPC/POPG lipid bilayers. The analogue peptide, MSI-584A, on the other hand, exhibited a larger bent angle between the N- (1-11) and C- (12-24) termini helices with the hydrophobic C-terminal helix inserted into the hydrophobic region of the bilayer (denoted "membrane-inserted") when interacting with both POPC and 7:3 POPC/POPG lipid bilayers. These experimental findings on the membrane orientations suggest that both peptides are likely to disrupt the cell membrane through the carpet mechanism.

Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a “transport” domain and a “scaffold” domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an ‘elevator-with-an-operator’ mechanism.

Emulation of the structure of the Saposin protein fold by a lung surfactant peptide construct of surfactant Protein B.

The three-dimensional structure of the synthetic lung Surfactant Protein B Peptide Super Mini-B was determined using an integrative experimental approach, including mass spectrometry and isotope enhanced Fourier-transform infrared (FTIR) spectroscopy. Mass spectral analysis of the peptide, oxidized by solvent assisted region-specific disulfide formation, confirmed that the correct folding and disulfide pairing could be facilitated using two different oxidative structure-promoting solvent systems. Residue specific analysis by isotope enhanced FTIR indicated that the N-terminal and C-terminal domains have well defined α-helical amino acid sequences. Using these experimentally derived measures of distance constraints and disulfide connectivity, the ensemble was further refined with molecular dynamics to provide a medium resolution, residue-specific structure for the peptide construct in a simulated synthetic lung surfactant lipid multilayer environment. The disulfide connectivity combined with the α-helical elements stabilize the peptide conformationally to form a helical hairpin structure that resembles critical elements of the Saposin protein fold of the predicted full-length Surfactant Protein B structure.

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.