Membrane-spanning Helices Research Articles

Unraveling the Membrane Topology of TMEM151A: A Step Towards Understanding its Cellular Role

Transmembrane protein 151A (TMEM151A) has been identified as a causative gene for paroxysmal kinesigenic dyskinesia, though its molecular function remains almost completely unknown. Understanding the membrane topology of transmembrane proteins is crucial for elucidating their functions and possible interacting partners. In this study, we utilized molecular dynamics simulations, immunocytochemistry, and electron microscopy to define the topology of TMEM151A. Our results validate a starting AlphaFold model of TMEM151A and reveal that it comprises a transmembrane domain with two membrane-spanning alpha helices connected by a short extracellular loop and an intramembrane helix-hinge-helix structure. Notably, most of the protein is oriented towards the intracellular side of the membranes with a large cytosolic domain featuring a combination of alpha-helix and beta-sheet structures, as well as the protein N- and C-termini. These insights into TMEM151A’s topology and orientation of its domains with respect of the cell membranes provide essential information for future functional studies and represent a first fundamental step for understanding its role in the pathogenesis of paroxysmal kinesigenic dyskinesia.

Identification of residues involved in homotrimeric assembly of the human P2X4 receptor by simulations and site-directed mutagenesis.

P2X receptors (P2XRs) are a family of ATP-gated cation channels with seven members, P2X1-P2X7. P2XRs are involved in numerous physiological and pathophysiological functions and are therefore interesting drug targets. Functional P2XRs are obligate homotrimers or heterotrimers of three identical or homologous subunits, respectively, with each subunit having a large ectodomain flanked by two membrane-spanning helices, TM1 and TM2, and N- and C-terminal intracellular domains.

Structure of the GOLD-domain seven-transmembrane helix protein family member TMEM87A.

TMEM87s are eukaryotic transmembrane proteins with two members (TMEM87A and TMEM87B) in humans. TMEM87s have proposed roles in protein transport to and from the Golgi, as mechanosensitive ion channels, and in developmental signaling. TMEM87 disruption has been implicated in cancers and developmental disorders. To better understand TMEM87 structure and function, we determined a cryo-EM structure of human TMEM87A in lipid nanodiscs. TMEM87A consists of a Golgi-dynamics (GOLD) domain atop a membrane-spanning seven-transmembrane helix domain with a large cavity open to solution and the membrane outer leaflet. Structural and functional analyses suggest TMEM87A may not function as an ion channel or G-protein coupled receptor. We find TMEM87A shares its characteristic domain arrangement with seven other proteins in humans; three that had been identified as evolutionary related (TMEM87B, GPR107, and GPR108) and four previously unrecognized homologs (GPR180, TMEM145, TMEM181, and WLS). Among these structurally related GOLD domain seven-transmembrane helix (GOST) proteins, WLS is best characterized as a membrane trafficking and secretion chaperone for lipidated Wnt signaling proteins. We find key structural determinants for WLS function are conserved in TMEM87A. We propose TMEM87A and structurally homologous GOST proteins could serve a common role in trafficking membrane-associated cargo.

Membrane Activity of a DNA-Based Ion Channel Depends on the Stability of Its Double-Stranded Structure.

DNA nanotechnology has emerged as a promising method for designing spontaneously inserting and fully controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based membrane channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA-lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications can cause fraying of terminal base pairs of nicked DNA constructs, distorting them when embedded in a lipid bilayer. Importantly, we show that DNA nanostructures with no backbone discontinuities form more stable conductive pores and insert into membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows design and maintenance of membrane-spanning helices in a tilted orientation within the lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells.

TMEM120A, a member of the transmembrane protein 120 (TMEM120) family, has a pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells and have solved cryo-electron microscopy (cryo-EM) structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing, or metabolism.

Determination of the membrane topology of PORCN, an O-acyl transferase that modifies Wnt signalling proteins.

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

SARS‐CoV‐2: pushing the E(nvelope)

SARS‐CoV‐2: pushing the E(nvelope)

NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4.

Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix-helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity.

TANGO1 membrane helices create a lipid diffusion barrier at curved membranes.

We have previously shown TANGO1 organises membranes at the interface of the endoplasmic reticulum (ER) and ERGIC/Golgi (Raote et al., 2018). TANGO1 corrals retrograde membranes at ER exit sites to create an export conduit. Here the retrograde membrane is, in itself, an anterograde carrier. This mode of forward transport necessitates a mechanism to prevent membrane mixing between ER and the retrograde membrane. TANGO1 has an unusual membrane helix organisation, composed of one membrane-spanning helix (TM) and another that penetrates the inner leaflet (IM). We have reconstituted these membrane helices in model membranes and shown that TM and IM together reduce the flow of lipids at a region of defined shape. We have also shown that the helices align TANGO1 around an ER exit site. We suggest this is a mechanism to prevent membrane mixing during TANGO1-mediated transfer of bulky secretory cargos from the ER to the ERGIC/Golgi via a tunnel.

Structure and mechanism of the ER-based glucosyltransferase ALG6.

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG2ALW5LALALALALALALW19LAG22A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state 2H NMR spectra of labeled alanine residues reveals marked differences with H2,22 compared to W2,22. Nevertheless, the properties of membrane-spanning H2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues.

Effect of pH and Lipid Composition on Membrane-Spanning Helices with Glutamic Acid Examined by Solid-State Nmr

Transmembrane proteins constitute about 30% of the proteins in the cell and are involved in major biological processes. The dynamic properties of membrane proteins and the ionization states of particular side chains are important for biological function. The biophysical properties of membrane proteins nevertheless can be difficult todecode, particularly for glutamic acid in the lipid environment of cell membranes (BBA Biomembranes 1859, 484). To study the ionization of glutamic acid in transmembrane peptides, guest glutamic acid residues were substituted into the well-defined model helix of GWALP23 (acetyl-GGAL4WLALALALALAL16ALWLAGA-amide).

Structures of the AMPA receptor in complex with its auxiliary subunit cornichon.

In the brain, AMPA-type glutamate receptors (AMPARs) form complexes with their auxiliary subunits and mediate the majority of fast excitatory neurotransmission. Signals transduced by these complexes are critical for synaptic plasticity, learning, and memory. The two major categories of AMPAR auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs); these subunits share little homology and play distinct roles in controlling ion channel gating and trafficking of AMPAR. Here, I report high-resolution cryo-electron microscopy structures of AMPAR in complex with CNIH3. Contrary to its predicted membrane topology, CNIH3 lacks an extracellular domain and instead contains four membrane-spanning helices. The protein-protein interaction interface that dictates channel modulation and the lipids surrounding the complex are revealed. These structures provide insights into the molecular mechanism for ion channel modulation and assembly of AMPAR/CNIH3 complexes.

Membrane Topological Model of Glycosyltransferases of the GT-C Superfamily.

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins

The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis. Here, we report a novel approach, site-directed alkylation detected by in-gel fluorescence (SDAF), which monitors the fluorescent band shift caused by alkylation of the EGFP-fused target membrane protein bearing one single introduced cysteine. In-gel fluorescence provides a unique readout of target membrane proteins with EGFP fusion from non-purified samples, revealing a distinct 5 kDa shift on SDS-PAGE gel due to conjugation with mPEG-MAL-5K. Using the structurally characterised bile acid transporter ASBTNM as an example, we demonstrate that SDAF generates a topology map consistent with the crystal structure. The efficiency of mPEG-MAL-5K modification at each introduced cysteine can easily be quantified and analysed, providing a useful tool for probing the solvent accessibility at a specific position of the target membrane protein.

Glutamate 95 in NqrE Is an Essential Residue for the Translocation of Cations in Na+-NQR.

The sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) is a bacterial enzyme that oxidizes NADH, reduces ubiquinone, and translocates Na+ across the membrane. We previously identified three acidic residues in the membrane-spanning helices, near the cytosol, NqrB-D397, NqrD-D133, and NqrE-E95, as candidates likely to be involved in Na+ uptake, and replacement of any one of them by a non-acidic residue affects the Na+-dependent kinetics of the enzyme. Here, we have inquired further into the role of the NqrE-E95 residue by constructing a series of mutants in which this residue is replaced by amino acids with charges and/or sizes different from those of the glutamate of the wild-type enzyme. All of the mutants showed altered steady-state kinetics with the acceleration of turnover by Na+ greatly diminished. Selected mutants were studied by other physical methods. Membrane potential measurements showed that NqrE-E95D and A are significantly less efficient in ion transport. NqrE-E95A, Q, and D were studied by transient kinetic measurements of the reduction of the enzyme by NADH. In all three cases, the results indicated inhibition of the electron-transfer step in which the FMNC becomes reduced. This is the first Na+-dependent step and is associated with Na+ uptake by the enzyme. Electrochemical measurements on NqrE-E95Q showed that the Na+ dependence of the redox potential of the FMN cofactors has been lost. The fact that the mutations at the NqrE-E95 site have specific effects related to translocation of Na+ and Li+ strongly indicates a definite role for NqrE-E95 in the cation transport process of Na+-NQR.

Lipid Optimization to Improve the Solid-State NMR Spectra from Membrane-Spanning Helices with Glutamic Acid

Lipid Optimization to Improve the Solid-State NMR Spectra from Membrane-Spanning Helices with Glutamic Acid

Structural and Functional Analyses of TRPC3 Reveal Allosteric Gating Modulation by the Cytoplasmic Domain

TRPC3 is associated with neurodegenerative diseases, memory loss, and hypertension. In blood vessels, TRPC3 is an essential component of the cellular mechanisms by which vasoconstrictors regulate blood pressure. Vasoconstrictors, such as angiotensin II, bind to phospholipase C (PLC)-coupled receptors, increasing the intracellular levels of DAG, which in turn activates TRPC3. Upon activation, TRPC3 channels induce depolarization increases intracellular Ca2+and promotes vasoconstriction. Here, we show the full-length and cytoplasmic domain (CPD) cryoEM structures for human TRPC3 in the apo state at 5.8 and 4.0 Å resolution. A TRPC3 subunit consists of an N-terminus formed by four ankyrin repeats, the linker helical domain (LHD1-9), and the pre-S1 elbow. The N-terminus is followed by six membrane-spanning helices (S1-S6) and a pore helix connecting to a re-entrant loop that forms the selectivity filter. The TRP helix is immediately adjacent to S6, and the C-terminus consists of two alpha-helices connected by a small linker. TRPC3 transmembrane domain resembles those of other TRP channels, whereas the CPD features major differences. A C-terminal domain swap occurs at the center of the CPD, where horizontal helices (HH) transition into a coiled-coil bundle, highlighting a unique fold for the TRPC subfamily. Comparison of TRPC3 structures reveals that the HH can reside in two distinct positions. Electrophysiological analyses demonstrate that TRPC3 activity increases by shortening the length of the C-terminal loop connecting the HH with the TRP helices; likewise elongating the length of the loop has the opposite effect. Our findings demonstrate that the C-terminal loop impacts channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. Future functional and structural experiments are underway to validate whether the upward movement of the HHs corresponds to a positive modulation of the TRPC3 gating cycle.

Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation.

The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation.IMPORTANCE G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system.

ONE-HELIX PROTEIN2 (OHP2) Is Required for the Stability of OHP1 and Assembly Factor HCF244 and Is Functionally Linked to PSII Biogenesis.

The members of the light-harvesting complex protein family, which include the one-helix proteins (OHPs), are characterized by one to four membrane-spanning helices. These proteins function in light absorption and energy dissipation, sensing light intensity, and triggering photomorphogenesis or the binding of chlorophyll and intermediates of chlorophyll biosynthesis. Arabidopsis (Arabidopsis thaliana) contains two OHPs, while four homologs (named high-light-induced proteins) exist in Synechocystis PCC6803. Various functions have been assigned to high-light-induced proteins, ranging from photoprotection and the assembly of photosystem I (PSI) and PSII to regulation of the early steps of chlorophyll biosynthesis, but little is known about the function of the two plant OHPs. Here, we show that the two Arabidopsis OHPs form heterodimers and that the stromal part of OHP2 interacts with the plastid-localized PSII assembly factor HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244). Moreover, concurrent accumulation of the two OHPs and HCF244 is critical for the stability of all three proteins. In particular, the absence of OHP2 leads to the complete loss of OHP1 and HCF244. We used a virus-induced gene silencing approach to minimize the expression of OHP1 or OHP2 in adult Arabidopsis plants and revealed that OHP2 is essential for the accumulation of the PSII core subunits, while the other photosynthetic complexes and the major light-harvesting complex proteins remained unaffected. We examined the potential functions of the OHP1-OHP2-HCF244 complex in the assembly and/or repair of PSII and propose a role for this heterotrimeric complex in thylakoid membrane biogenesis.