Α-helical Transmembrane Segments Research Articles

Expression, Purification and Characterization of a ZIP Family Transporter from Desulfovibrio vulgaris.

The ZIP family transport zinc ions from the extracellular medium across the plasma membrane or from the intracellular compartments across endomembranes, which play fundamental roles in metal homeostasis and are broadly involved in physiological and pathological processes. Desulfovibrio is the predominant sulphate-reducing bacteria in human colonic microbiota, but also a potential choice for metal bioremediation. while, there are no published studies describing the zinc transporters from Desulfovibrio up to now. In this study, we obtained for the first time a heterologously expressed ZIP homolog from Desulfovibrio vulgaris, termed dvZip. The purified dvZip was reconstituted into proteoliposomes, and confirmed its zinc transport ability in vitro. By combining topology prediction, homology modeling and phylogenetic approaches, we also noticed that dvZip belongs to the GufA and probably have 8 transmembrane α-helical segments (TM 1-8) in which both termini are located on the extracellular, with TM2, 4, 5 and 7 create an inner bundle. We believe that purification and characterization of zinc (probably also cadmium) transporters from Desulfovibrio vulgaris such as dvZip could shed light on understanding of metal homeostasis of Desulfovibrio and provided protein products for future detailed function and structural studies.

Effects of intersubunit amino acid substitutions on GABA receptor sensitivity to the ectoparasiticide fluralaner

The isoxazoline ectoparasiticide fluralaner exerts antiparasitic effects by inhibiting the function of γ-aminobutyric acid (GABA) receptors (GABARs). The present study was conducted to identify the amino acid residues that contribute to the high sensitivity of insect GABARs to fluralaner. We generated housefly (Musca domestica) GABARs with amino acid substitutions in the first through third α-helical transmembrane segments (TM1–TM3) of the RDL subunit using site-directed mutagenesis and examined the effects of the substitutions on the sensitivity of GABARs expressed in Xenopus oocytes to fluralaner using two-electrode voltage clamp electrophysiology. The Q271L substitution in TM1 caused a significant reduction in the sensitivity to fluralaner. Although the I274A and I274F substitutions in TM1 did not affect fluralaner sensitivity, the I274C substitution significantly enhanced the sensitivity to fluralaner. In contrast, the L278C substitution in TM1 reduced fluralaner sensitivity. Substitutions of Gly333 in TM3 led to substantial reductions in the sensitivity to fluralaner. These findings indicate that Gln271, Ile274, Leu278, and Gly333, which are situated in the outer half of the transmembrane subunit interface, are closely related to the antagonism of GABARs by fluralaner.

Membrane Protein Integration and Topogenesis at the ER.

Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished-the signal, the stop-transfer sequence, and the re-integration sequence-which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones.

Role of the signal sequence in proteorhodopsin biogenesis in E. coli.

Blue-absorbing proteorhodopsin (BPR) from marine bacteria is a retinal-bound, light-activated, outwards proton transporter containing seven α-helical transmembrane segments (TMS). It is synthesized as a precursor species (pre-BPR) with a predicted N-terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre-BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre-BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild-type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial "pro" domain rather than a traditional secretory peptide.

Alternative dimerization of receptor tyrosine kinases with signal transduction through a cellular membrane

Receptor tyrosine kinases (RTKs) occupy a separate functional niche among membrane receptors, which is determined by the special features of mechanisms of the signal transduction through a cellular membrane. RTKs are involved in the regulation of development and homeostasis of all the tissues of a human organism, playing a central role in cell proliferation, differentiation, and adhesion. A necessary condition of the biochemical signal transduction through a plasmatic membrane is a ligand-dependent or a ligand-independent dimerization (and/or an oligomerization) of RTKs which is accompanied by conformational rearrangements of all the RTK domains, including the α-helical transmembrane segments. In this review, the main aspects of structure-function relationship for RTKs from various receptor subfamilies are briefly discussed. It is shown in the light of the recently obtained biophysical and biochemical data that functioning of RTK receptors is mediated not only by protein–protein interactions, but by the state of the lipid environment as one of the main components of a self-consistent signal transduction system as well. The new principles of intercellular signal transduction through a membrane replenish the molecular mechanisms of the RTK functioning that have been earlier proposed and explain a number of paradoxes which are observed upon activation of wild-type receptors and the receptors with pathogenic transmembrane mutations. Understanding of the complex mechanisms of the signaling processes can facilitate the successful search for new opportunities of influence on the RTK biological functions with potential therapeutic consequences.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.

Topological and phylogenetic analyses of bacterial holin families and superfamilies

Holins are small “hole-forming” transmembrane proteins that mediate bacterial cell lysis during programmed cell death or following phage infection. We have identified fifty two families of established or putative holins and have included representative members of these proteins in the Transporter Classification Database (TCDB; www.tcdb.org). We have identified the organismal sources of members of these families, calculated their average protein sizes, estimated their topologies and determined their relative family sizes. Topological analyses suggest that these proteins can have 1, 2, 3 or 4 transmembrane α-helical segments (TMSs), and members of a single family are frequently, but not always, of a single topology. In one case, proteins of a family proved to have either 2 or 4 TMSs, and the latter arose by intragenic duplication of a primordial 2 TMS protein-encoding gene resembling the former. Using established statistical approaches, some of these families have been shown to be related by common descent. Seven superfamilies, including 21 of the 52 recognized families were identified. Conserved motif and Pfam analyses confirmed most superfamily assignments. These results serve to expand upon the scope of channel-forming bacterial holins.

Topological mapping methods for α-helical bacterial membrane proteins--an update and a guide.

Integral membrane proteins with α-helical transmembrane segments (TMS) are known to play important and diverse roles in prokaryotic cell physiology. The net hydrophobicity of TMS directly corresponds to the observed difficulties in expressing and purifying these proteins, let alone producing sufficient yields for structural studies using two-/three-dimensional (2D/3D) crystallographic or nuclear magnetic resonance methods. To gain insight into the function of these integral membrane proteins, topological mapping has become an important tool to identify exposed and membrane-embedded protein domains. This approach has led to the discovery of protein tracts of functional importance and to the proposition of novel mechanistic hypotheses. In this review, we synthesize the various methods available for topological mapping of α-helical integral membrane proteins to provide investigators with a comprehensive reference for choosing techniques suited to their particular topological queries and available resources.

Wzx flippase‐mediated membrane translocation of sugar polymer precursors in bacteria

Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 α-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration

Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane α-helical segments in the nascent chain exit the translocon through a lateral gate that opens toward the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration.

Modeling, Substrate Docking, and Mutational Analysis Identify Residues Essential for the Function and Specificity of a Eukaryotic Purine-Cytosine NCS1 Transporter

The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a three-dimensional model of the Aspergillus nidulans purine-cytosine/H(+) FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1-5 and TMS6-10) that are followed by two additional TMSs. TMS1, TMS3, TMS6, and TMS8 form an open cavity that is predicted to host the substrate binding site. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; Ser-85 (TMS1), Trp-159, Asn-163 (TMS3), Trp-259 (TMS6), and Asn-354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus Asn-350, Asn-351, and Pro-353 are irreplaceable for FcyB function. Among these residues, Ser-85, Asn-163, Asn-350, Asn-351, and Asn-354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that Ser-85, Asn-163, and Asn-354 directly interact with substrates, Trp-159 and Trp-259 stabilize binding through π-π stacking interactions, and Pro-353 affects the local architecture of substrate binding site, whereas Asn-350 and Asn-351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family by combining genetic and computational approaches.

Mapping out forces that act on transmembrane helices during membrane insertion

Membrane proteins destined for insertion into the inner membrane of E. coli are synthesized by ribosomes bound to the SecYEG translocon. During co‐translational membrane integration, transmembrane α‐helical segments in the nascent chain exit the translocon through a lateral gate that opens towards the surrounding membrane, but the mechanism of gate opening and lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when inside the translocon. Current work in our lab has shown that a force proportional to the free energy of membrane insertion is exerted on a transmembrane helix when it transits through the translocon channel, providing a direct insight into the dynamics of membrane integration.

The Amino Acid-Polyamine-Organocation Superfamily

The amino acid-polyamine-organocation (APC) superfamily has been shown to include five recognized families, four of which are specific for amino acids and their derivatives. Recent high-resolution X-ray crystallographic data have shown that four additional transporter families (BCCT, TC No. 2.A.15; SSS, 2.A.21; NSS, 2.A.22; and NCS1, 2.A.39), transporting a wide range of solutes, exhibit sufficiently similar folds to suggest a common evolutionary origin. We have used established statistical methods, based on sequence similarity, to show that these families are, in fact, members of the APC superfamily. We also identify two additional families (NCS2, 2.A.40; SulP, 2.A.53) as being members of this superfamily. Repeat sequences, each having five transmembrane α-helical segments and arising via ancient intragenic duplications, are demonstrated for all of these families, further strengthening the conclusion of homology. The APC superfamily appears to be the second largest superfamily of secondary carriers, the largest being the major facilitator superfamily (MFS). Although the topology of the members of the APC superfamily differs from that of the MFS, both families appear to have arisen from a common ancestral 2 TMS hairpin structure that underwent intragenic triplication followed by loss of a TMS in the APC family, to give the repeat units that are characteristic of these two superfamilies.

Thermodynamic Approach to Large-Scale Modeling of Alpha-Helices in Membranes

A theoretical approach to large-scale modeling of α-helices in micelles and lipid bilayers has been developed. The FMAP (Folding of Membrane-Associated Peptides) method identifies potential α-helices in amino acid sequences of peptides and proteins, calculates their thermodynamic stability and membrane-binding affinity, and provides full-atomic 3D models arranged with respect to membranes. The method accounts for: (a) backbone enthalpic and entropic contributions and side chain secondary structure propensities, as described by a thermodynamic model of helix-coil transition (Framework1); (b) transfer free energy of α-helical and coil segments from water to anisotropic membrane environment (including the long-range electrostatics and first-shell solvation components), as described by implicit solvent model of the lipid bilayer (PPM2); and (c) van der Waals interactions and H-bonds between side chains, as defined by interatomic potentials derived from protein engineering data3,4. FMAP was thoroughly tested for more than a hundred of experimentally studied peptides. It was subsequently applied for modeling of transmembrane α-helical segments in ∼2500 single-spanning proteins from the entire human proteome. The results of the calculations are available in the Membranome database at membranome.org.1. Lomize AL, Mosberg HI. Thermodynamic model of secondary structure for alpha-helical peptides and proteins. Biopolymers 1997, 42:239-269.2. Lomize AL, Pogozheva ID, Mosberg HI. Anisotropic solvent model of the lipid bilayer. 2. Energetics of insertion of small molecules, peptides, and proteins in membranes. J Chem Inf Model 2011, 51:930-946.3. Lomize AL, Reibarkh MY, Pogozheva ID. Interatomic potentials and solvation parameters from protein engineering data for buried residues. Protein Sci 2002, 11:1984-2000.4. Lomize AL, Pogozheva ID, Mosberg HI. Quantification of helix-helix binding affinities in micelles and lipid bilayers. Protein Sci 2004, 13:2600-2612

Engineering a Prokaryotic Cys-loop Receptor with a Third Functional Domain

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.

Structure and dynamics of the lipid modifications of a transmembrane α-helical peptide determined by 2H solid-state NMR spectroscopy

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the 2H and 31P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant.

Biogenesis of Mitochondria: Dual Role of Tom7 in Modulating Assembly of the Preprotein Translocase of the Outer Membrane

Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central β-barrel forming protein Tom40 with six different subunits that are embedded in the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of β-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM–Mdm10 machinery; however, different views exist on the function of the SAM–Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM–Mdm10 complex. Since the SAM–Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the β-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.

Assembly of the Mitochondrial Protein Import Channel

The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.