Site-directed spin labeling of a bacterial chemoreceptor reveals a dynamic, loosely packed transmembrane domain

We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to investigate dynamics and helical packing in the four-helix transmembrane domain of the homodimeric bacterial chemoreceptor Trg. We focused on the first transmembrane helix, TM1, particularly on the nine-residue sequence nearest the periplasm, because patterns of disulfide formation between introduced cysteines had identified that segment as the region of closest approach among neighboring transmembrane helices. Along this sequence, mobility and accessibility of the introduced spin label were characteristic of loosely packed or solvent-exposed side chains. This was also the case for eight additional positions around the circumference and along the length of TM1. For the continuous nine-residue sequence near the periplasm, mobility and accessibility varied only modestly as a function of position. We conclude that side chains of TM1 that face the interior of the four-helix domain interact with neighboring helices but dynamic movement results in loose packing. Compared to transmembrane segments of other membrane proteins reconstituted into lipid bilayers and characterized by site-directed spin labeling, TM1 of chemoreceptor Trg is the most dynamic and loosely packed. A dynamic, loosely packed chemoreceptor domain can account for many experimental observations about the transmembrane domains of chemoreceptors.     

Production and initial structural characterization of the TM4TM5 helix‐loop‐helix domain of the translocator protein

Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix–helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C‐terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far‐UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n‐dodecyl‐β‐d ‐maltoside, n‐octyl‐β‐d ‐glucoside, n‐dodecylphosphocholine, 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC), and 1‐palmitoyl‐2‐hydroxy‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5‐DHPC complex. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core

The folding of most integral membrane proteins follows a two‐step process: initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four‐helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step, this intermediate is folded into a six‐helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: first, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3, and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD‐simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process.     

A high resolution structure of the putative hinge region in M2 channel-lining segments of the nicotinic acetylcholine receptor

Transmembrane peptide helices play key roles in signal transduction across cell membranes, yet little is known about their high-resolution structure or the role membrane composition plays in their association, structure, dynamics and ultimately their performance. Using magic angle spinning (MAS) homonuclear dipolar recoupling experiments, the backbone structure at positions L10, L11, and A12 of the M2 ion channel peptide was determined in two lipid systems. Their measurements are in agreement with M2 forming transmembrane helices, but the torsion angles vary considerably from common α−helical values. These measurements show remarkable agreement with a previous computational model of M2 peptides forming a pore domain in which their helices are kinked near the central leucine, L11 [R. Sankararamakrishnan, C. Adcock, M.S.P. Sansom, The pore domain of the nicotinic acetylcholine receptor: Molecular modeling, pore dimensions, and electrostatics, Biophys. J. 71 (1996) 1659-1671]. The generation of high resolution data for transmembrane helices is of critical importance in refining structures for membrane protein and developing models of helix packing interactions.     

Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand‐recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence‐divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg_E from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross‐linking between pairs of introduced cysteines. A homology model of the Trg_E domain was created using the homodimeric, four‐helix bundle structure of the Tar_S domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg_E periplasmic domain is very similar to the Tar_S domain. Diagnostic cross‐linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.     

Bilayer mechanical properties regulate the transmembrane helix mobility and enzymatic state of CD39

CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In this study, we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics. Alteration of membrane properties by insertion of cone-shaped or inverse cone-shaped amphiphiles or by cholesterol removal switches CD39 to the same enzymatic state that removal or solubilization of the transmembrane domains does. The same membrane alterations increase the propensity of both transmembrane helices to rotate within the packed structure, resulting in a structure with greater mobility but not an altered primary conformation. Membrane alteration also abolishes the ability of the substrate to stabilize the helices in their primary conformation, indicating a loss of coupling between substrate binding and transmembrane helix dynamics. Removal of either transmembrane helix mimics the effect of membrane alteration on the mobility and substrate sensitivity of the remaining helix, suggesting that the ends of the extracellular domain have intrinsic flexibility. We suggest that a mechanical bilayer property, potentially elasticity, regulates CD39 by altering the balance between the stability and flexibility of its transmembrane helices and, in turn, of its active site.     

Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Influence of Membrane Lipid Composition on a Transmembrane Bacterial Chemoreceptor

Most bacterial chemoreceptors are transmembrane proteins. Although less than 10% of a transmembrane chemoreceptor is embedded in lipid, separation from the natural membrane environment by detergent solubilization eliminates most receptor activities, presumably because receptor structure is perturbed. Reincorporation into a lipid bilayer can restore these activities and thus functionally native structure. However, the extent to which specific lipid features are important for effective restoration is unknown. Thus we investigated effects of membrane lipid composition on chemoreceptor Tar from Escherichia coli using Nanodiscs, small (∼10-nm) plugs of lipid bilayer rendered water-soluble by an annulus of “membrane scaffold protein.” Disc-enclosed bilayers can be made with different lipids or lipid combinations. Nanodiscs carrying an inserted receptor dimer have high protein-to-lipid ratios approximating native membranes and in this way mimic the natural chemoreceptor environment. To identify features important for functionally native receptor structure, we made Nanodiscs using natural and synthetic lipids, assaying extents and rates of adaptational modification. The proportion of functionally native Tar was highest in bilayers closest in composition to E. coli cytoplasmic membrane. Some other lipid compositions resulted in a significant proportion of functionally native receptor, but simply surrounding the chemoreceptor transmembrane segment with a lipid bilayer was not sufficient. Membranes effective in supporting functionally native Tar contained as the majority lipid phosphatidylethanolamine or a related zwitterionic lipid plus a rather specific proportion of anionic lipids, as well as unsaturated fatty acids. Thus the chemoreceptor is strongly influenced by its lipid environment and is tuned to its natural one.     

Molecular dynamics simulation of human immunodeficiency virus protein U (Vpu) in lipid/water Langmuir monolayer

Virus protein U (Vpu) is an accessory membrane protein encoded by human immunodeficiency virus type 1 (HIV-1). Various NMR and CD studies have shown that the transmembrane domain of Vpu has a helical conformation and that the cytoplasmic domain adopts the helix-loop-helix-turn motif. This 3.5-ns molecular dynamics (MD) simulation of Vpu in a lipid/membrane environment has fully reproduced these structural characteristics. Membrane propensities of two amphipathic helices in the cytoplasmic domain are further compared here to understand better their complicated orientational behavior known from experiment. This study first reveals that the highly conserved loop region in the cytoplasmic domain can be closely associated with the membrane surface. It is known from the simulation that Vpu is associated with 34 lipids in this Langmuir monolayer. The lipids that are located between the Vpu transmembrane helix and the first helix in the cytoplasmic domain are pushed up by Vpu. These elevated lipids have increased P-N tilt angles for the head groups but unchanged acyl-chain tilt angles compared with lipids that do not interact with Vpu. This study verifies the significance of applying MD simulation in refining protein structure and revealing detailed protein-lipid interaction in membrane/water environment. Figure XZ view of a snapshot of Vpu/DLGPC/water system after 3.5 ns NP(N)gamma T MD simulation. Coloring scheme: Vpu, red; C, green; H, pink; N, blue; O, orange; P, magenta; water, light blue     

Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix

The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins.     

Two Cooperating Helices Constitute the Lipid-binding Domain of the Bacterial SRP Receptor

Protein targeting by the bacterial signal recognition particle requires the specific interaction of the signal recognition particle (SRP)-ribosome-nascent chain complex with FtsY, the bacterial SRP receptor. Although FtsY in Escherichia coli lacks a transmembrane domain, the membrane-bound FtsY displays many features of an integral membrane protein. Our data reveal that it is the cooperative action of two lipid-binding helices that allows this unusually strong membrane contact. Helix I comprises the first 14 amino acids of FtsY and the second is located at the interface between the A- and the N-domain of FtsY. We show by site-directed cross-linking and binding assays that both helices bind to negatively charged phospholipids, with a preference for phosphatidyl glycerol. Despite the strong lipid binding, helix I does not seem to be completely inserted into the lipid phase, but appears to be oriented parallel with the membrane surface. The two helices together with the connecting linker constitute an independently folded domain, which maintains its lipid binding even in the absence of the conserved NG-core of FtsY. In summary, our data reveal that the two consecutive lipid-binding helices of FtsY can provide a membrane contact that does not differ significantly in stability from that provided by a transmembrane domain. This explains why the bacterial SRP receptor does not require an integral β-subunit for membrane binding.     

Stabilization of conformationally dynamic helices by covalently attached acyl chains

Acylation of proteins is known to mediate membrane attachment and to influence subcellular sorting. Here, we report that acylation can stabilize secondary structure. Circular dichroism spectroscopy showed that N‐terminal attachment of acyl chains decreases the ability of an intrinsically flexible hydrophobic model peptide to refold from an α‐helical state to β‐sheet in response to changing solvent conditions. Acylation also stabilized the membrane‐embedded α‐helix. This increase of global helix stability did not result from decreased local conformational dynamics of the helix backbone as assessed by deuterium/hydrogen‐exchange experiments. We concluded that acylation can stabilize the structure of intrinsically dynamic helices and may thus prevent misfolding.     

Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain.

The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.     

Phosphorylation stabilizes the N‐termini of α‐helices

The role of phosphorylation in stabilizing the N‐termini of α‐helices is examined using computer simulations of model peptides. The models comprise either a phosphorylated or unphosphorylated serine at the helix N‐terminus, followed by nine alanines. Monte Carlo/stochastic Dynamics simulations were performed on the model helices. The simulations revealed a distinct stabilization of the helical conformation at the N‐terminus after phosphorylation. The stabilization was attributable to favorable electrostatic interactions between the phosphate and the helix backbone. However, direct helix capping by the phosphorylated sidechain was not observed. The results of the calculations are consistent with experimental evidence on the stabilization of helices by phosphates and other anions. © 1999 John Wiley & Sons, Inc. Biopoly 49: 225–233, 1999     

Structure prediction of the dimeric neu/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations

Dimerization of the neu/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of the helical transmembrane domain (TM) of neu and ErbB-2 receptors are used to predict their dimer structure both in the wild and oncogenic forms. A global conformational search method, applied to define the best orientations of parallel helices, showed an energetically favorable configuration with the specific mutation site within the interface, common for both the nontransforming and the transforming neu/ErbB-2 TM dimers. Starting from this configuration, a total of 10 simulations, about 1.4 ns each, performed in vacuum, without any constraints, show that the two helices preferentially wrap in left-handed interactions with a packing angle at about 20°. The resulting structures are nonsymmetric and the hydrogen bond network analysis shows that helices experience π local distortions that facilitate inter-helix hydrogen bond interactions and may result in a change in the helix packing, leading to a symmetric interface. For the mutated sequences, we show that the Glu side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. We show that the connectivity between interfacial residues conforms to the knobs-into-holes packing mode of transmembrane helices. The dimeric interface described in our models is discussed with respect to mutagenesis studies. Received: 12 March 1999 / Revised version: 23 August 1999 / Accepted: 23 August 1999     

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor,a human GPCR

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)₆ tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.     

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic-aqueous medium

The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.     

Structure of the hypothetical protein Ton 1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left‐handed helical turn in normal α‐solenoid protein

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α‐helices in a helix‐turn‐helix motif as a repeating unit, Ton1535 consists of right‐handed coiled N‐ and C‐terminal regions that are stacked together using helix bundles containing a left‐handed helical turn. One left‐handed helical turn in the right‐handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α‐helices on the convex surfaces of the N‐terminal region to the extended surface of the concave groove of the C‐terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.     

Simulation of voltage-dependent interactions of α-helical peptides with lipid bilayers

Pore formation in lipid bilayers by channel-forming peptides and toxins is thought to follow voltage-dependent insertion of amphipathic α-helices into lipid bilayers. We have developed an approximate potential for use within the CHARMm molecular mechanics program which enables one to simulate voltage-dependent interaction of such helices with a lipid bilayer. Two classes of helical peptides which interact with lipid bilayers have been studied: (a) δ-toxin, a 26 residue channel-forming peptide from Staphylococcus aureus; and (b) synthetic peptides corresponding to the α5 and α7 helices of the pore-forming domain of Bacillus thuringiensis CryIIIA δ-endotoxin. Analysis of δ-toxin molecular dynamics (MD) simulations suggested that the presence of a transbilayer voltage stabilized the inserted location of δ-toxin helices, but did not cause insertion per se. A series of simulations for the α5 and α7 peptides revealed dynamic switching of the α5 helix between a membrane-associated and a membrane-inserted state in response to a transbilayer voltage. In contrast the α7 helix did not exhibit such switching but instead retained a membrane associated state. These results are in agreement with recent experimental studies of the interactions of synthetic α5 and α7 peptides with lipid bilayers.