NMR structure of the transmembrane domain of the n-acetylcholine receptor β2 subunit

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.     

Improving the detection of transmembrane β-barrel chains with N-to-1 extreme learning machines

MOTIVATION: Transmembrane β-barrels (TMBBs) are extremely important proteins that play key roles in several cell functions. They cross the lipid bilayer with β-barrel structures. TMBBs are presently found in the outer membranes of Gram-negative bacteria and of mitochondria and chloroplasts. Loop exposure outside the bacterial cell membranes makes TMBBs important targets for vaccine or drug therapies. In genomes, they are not highly represented and are difficult to identify with experimental approaches. Several computational methods have been developed to discriminate TMBBs from other types of proteins. However, the best performing approaches have a high fraction of false positive predictions. RESULTS: In this article, we introduce a new machine learning approach for TMBB detection based on N-to-1 Extreme Learning Machines that significantly outperforms previous methods achieving a Matthews correlation coefficient of 0.82, a probability of correct prediction of 0.92 and a sensitivity of 0.73.     

Inward-facing glycine residues create sharp turns in β-barrel membrane proteins

The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively β-barrels composed of between 8 and 26 β-strands. To explore the relationship between β-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 β-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the β-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in β-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in β-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Pattern of amino acid substitutions in transmembrane domains of β-barrel membrane proteins for detecting remote homologs in bacteria and mitochondria

β-barrel membrane proteins play an important role in controlling the exchange and transport of ions and organic molecules across bacterial and mitochondrial outer membranes. They are also major regulators of apoptosis and are important determinants of bacterial virulence. In contrast to β-helical membrane proteins, their evolutionary pattern of residue substitutions has not been quantified, and there are no scoring matrices appropriate for their detection through sequence alignment. Using a Bayesian Monte Carlo estimator, we have calculated the instantaneous substitution rates of transmembrane domains of bacterial β-barrel membrane proteins. The scoring matrices constructed from the estimated rates, called bbTM for β-barrel Transmembrane Matrices, improve significantly the sensitivity in detecting homologs of β-barrel membrane proteins, while avoiding erroneous selection of both soluble proteins and other membrane proteins of similar composition. The estimated evolutionary patterns are general and can detect β-barrel membrane proteins very remote from those used for substitution rate estimation. Furthermore, despite the separation of 2-3 billion years since the proto-mitochondrion entered the proto-eukaryotic cell, mitochondria outer membrane proteins in eukaryotes can also be detected accurately using these scoring matrices derived from bacteria. This is consistent with the suggestion that there is no eukaryote-specific signals for translocation. With these matrices, remote homologs of β-barrel membrane proteins with known structures can be reliably detected at genome scale, allowing construction of high quality structural models of their transmembrane domains, at the rate of 131 structures per template protein. The scoring matrices will be useful for identification, classification, and functional inference of membrane proteins from genome and metagenome sequencing projects. The estimated substitution pattern will also help to identify key elements important for the structural and functional integrity of β-barrel membrane proteins, and will aid in the design of mutagenesis studies.     

The Monomer-Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.     

NMR structures of the transmembrane domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.     

BamE structure: the assembly of β-barrel proteins in the outer membranes of bacteria and mitochondria

A study recently published in EMBO reports solves the solution structure of E. coli BamE, thus providing the basis for a better understanding of the mechanism of β-barrel assembly in bacterial and mitochondrial outer membranes.EMBO Rep (2011) advance online publication. doi: 10.1038/embor.2010.202β-barrel membrane proteins are found exclusively in the outer membrane of Gram-negative bacteria and the outer membranes of eukaryotic organelles of prokaryotic origin, mitochondria and chloroplasts. In contrast to the inner membrane, the bacterial outer membrane is an asymmetrical bilayer that consists mainly of lipopolysaccharides in the outer leaflet and phospholipids in the inner leaflet. Bacterial β-barrel outer membrane proteins (OMPs) mediate many cellular functions, for example, passive or selective diffusion of small molecules through the β-barrel pores across the outer membrane. By contrast, only a few mitochondrial β-barrel outer membrane proteins (MBOMPs) have been identified so far. The central machineries that mediate insertion and assembly of OMPs/MBOMPs are the β-barrel assembly machine (BAM) complex in the bacterial outer membrane and the topogenesis of outer-membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex in the mitochondrial outer membrane (Knowles et al, 2009; Endo & Yamano, 2010; Stroud et al, 2010; Fig 1). However, the molecular mechanisms of β-barrel protein topogenesis in bacterial and mitochondrial outer membranes remain poorly understood.Open in a separate windowFigure 1β-barrel protein assembly in bacterial and mitochondrial outer membranes. (A) Bacteria. Ribbon models of the structures of the Sec complex, SurA, BamA (Clantin et al, 2007; Kim et al, 2007), BamE and OMP. The upper and lower inserts show the surface of BamE (residues 20–108; viewed after approximately 90° rotation of the ribbon model around the horizontal axis toward the reader). Residues important for BamD binding are shown in red and residues with NMR signals that were perturbed by BamD binding are shown in yellow. The residue (Phe 74) important for PG binding is shown in red and the residues with NMR signals that were perturbed by PG binding are shown in yellow. (B) Mitochondria. Ribbon models were drawn for the structures of small Tim and MBOMP. IM, inner membrane; IMS, intermembrane space; MBOMP, mitochondrial β-barrel outer membrane protein; OM, outer membrane; OMP, outer membrane protein; PG, phosphatidylglycerol; POTRA, polypeptide transport-associated domain.Bacterial OMPs are synthesized in the cytosol as precursor proteins with an amino-terminal signal sequence that guides the proteins to the Sec machinery for crossing the inner membrane and is cleaved off in the periplasm. Periplasmic chaperones then escort OMPs through the aqueous periplasmic space in a partly unfolded state. On reaching the outer membrane, OMPs assemble into a β-barrel structure and insert into the outer membrane with the help of the BAM complex. The bacterial OMP insertion pathway can be compared to the assembly pathway of MBOMPs from the mitochondrial intermembrane space into the outer membrane. MBOMPs are synthesized in the cytosol and imported into the intermembrane space by the outer membrane translocator TOM40. The subsequent chaperone-mediated escort across the intermembrane space and insertion into the outer membrane by the TOB complex is similar to the OMP assembly process. Notably, the BAM and TOB complexes share the homologous β-barrel proteins BamA and Tob55/Sam50, respectively, as the central components of their insertion machineries. The BAM complex in Escherichia coli consists of BamA (YaeT/Omp85) and four accessory lipoproteins: BamB (YfgL), BamC (NlpB), BamD (YfiO) and BamE (SmpA). BamA and BamD are essential for cell growth, yet deletion of dispensable BamB, BamC or BamE leads to outer membrane defects manifested in hypersensitivity to antibiotics. Although BamAB and BamCDE can form distinct subcomplexes, they become functional only after formation of the entire BAM complex with all five subunits (Hagan et al, 2010).In this issue of EMBO reports, Knowles et al (2011) solve the nuclear magnetic resonance (NMR) solution structure of E. coli BamE, which sheds light on the roles of one of the Bam subunits in β-barrel protein assembly. The structure of BamE consists of a three-stranded antiparallel β-sheet packed against a pair of α-helices (Fig 1).As the ΔbamE mutant cannot grow in the presence of vancomycin, the authors identify functionally important residues of BamE by testing the effects of amino-acid substitutions in BamE on its inability to complement the growth defects of ΔbamE, without destabilizing BamE itself. Many of the identified residues are conserved among BamE proteins from different organisms and map to a single surface area on BamE. Interestingly, NMR signals of the residues around this region are sensitive to the addition of micelles containing the lipid phosphatidylglycerol, but not phosphatidylethanolamine or cardiolipin. In parallel, the authors analyse perturbation of the NMR spectra of BamE after the addition of purified BamB, C and D proteins. Only BamD affects the NMR spectra of BamE, and the BamD interacting region of BamE is found to overlap partly with the residues involved in phosphatidylglycerol binding. As the addition of BamD and phosphatidylglycerol have different effects on the NMR spectra of BamE, the binding of BamD and phosphatidylglycerol to BamE seem to take place simultaneously. What is the biological relevance of the observed interactions of BamE with both BamD and phosphatidylglycerol? As phosphatidylglycerol was found to help the insertion of OMPs into lipid liposomes (Patel et al, 2009), BamE might recruit the BAM complex through BamD to phosphatidylglycerol-rich regions in the outer membrane, or might directly recruit phosphatidylglycerol to form assembly points for OMP insertion and folding.What are the roles of other subunits of the BAM complex in β-barrel protein assembly? The essential subunit of the E. coli BAM complex BamA consists of two domains: the N-terminal polypeptide transport-associated (POTRA) domain repeat in the periplasm and the carboxy-terminal β-barrel domain, embedded in the outer membrane. The number of POTRA domains ranges from one to five in BamA homologues from different organisms. Of these POTRA domains, the one nearest to the C-terminal that is most connected to the β-barrel domain is essential for cell viability and its deletion leads to disassembly of the BAM complex (Kim et al, 2007). Structural studies of the E. coli BamA POTRA domains suggest that each POTRA domain has a common fold, whereas conformational rigidity might differ between inter-domain linkers (Gatzeva-Topalova et al, 2010; Fig 1). As individual POTRA domains have some affinity for unfolded substrate proteins, the periplasmic tandem POTRA repeat probably provides several substrate binding sites that slide the substrate progressively towards the BamA β-barrel domain. The β-barrel domain of BamA probably functions as a scaffold to facilitate the formation of β-strands, possibly through β-augmentation and subsequent spontaneous membrane insertion of the β-barrel. Yet, it is not clear whether this cradle for β-strand formation is provided by the pore formed within the monomer or oligomeric forms of the BamA β-barrel domain. Alternatively, membrane insertion and folding of OMPs might take place at the interface between BamA and the outer membrane lipid bilayer.How much of the β-barrel assembly process is conserved during the evolution of mitochondria from Gram-negative bacteria? Although the central subunits BamA and Tob55 of the BAM and TOB complexes are conserved, other subunits of these complexes are unrelated to each other. The POTRA domains of BamA are essential for recognition and assembly of bacterial OMPs, whereas that of Tob55 is dispensable for MBOMP assembly in the mitochondrial outer membrane. Nevertheless, the mitochondrial TOB complex facilitates assembly of bacterial OMPs at low efficiency (Walther et al, 2009) and, in turn, the bacterial BAM complex can mediate assembly of mitochondrial porin. Therefore, the basic mechanism of β-barrel assembly in the outer membranes of bacteria and mitochondria seems to be conserved. High-resolution structures of each component of the BAM and TOB complexes—including that of BamE in this study—will thus provide the basis for a better understanding of the mechanism of β-barrel assembly in evolutionarily related bacterial and mitochondrial outer membranes.     

Polymerization of β-amino Acids in Aqueous Solution

We have compared carbonyl diimidazole (CDI) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as activating agents for the oligomerization of negatively-charged - and -amino acids in homogeneous aqueous solution. -Amino acids can be oligomerized efficiently using CDI, but not by EDAC. -Amino acids can be oligomerized efficiently using EDAC, but not by CDI. Aspartic acid, an - and -dicarboxylic acid is oligomerized efficiently by both reagents. These results are explained in terms of the mechanisms of the reactions, and their relevance to prebiotic chemistry is discussed.     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

Physical determinants of β-barrel membrane protein folding in lipid vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)(60) and Opa(50) into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Physical mapping of inhibin β-A in domestic cattle

The gene for the -A subunit of inhibin (INHBA) was assigned to bovine syntenic group U13 by bovine x rodent hybrid somatic cells and the polymerase chain reaction (PCR). A 482-bp PCR fragment was used to clone a 37-kb cosmid. This cosmid was assigned to bovine Chromosome (Chr) 4 (BTA 4) by fluorescence in situ hybridization (FISH). This is the first assignment of a U13 marker to a bovine chromosome. A restriction fragment length polymorphism (RFLP) was detected with PstI within the INHBA cosmid.     

Structural plasticity in self-assembling transmembrane β-sheets

Here we test the hypothesis that membrane-spanning β-sheets can exhibit structural plasticity in membranes due to their ability to shift hydrogen-bonding patterns. Transmembrane β-sheet forming peptides of the sequence AcWL_n, where n = 5, 6, or 7, which range from 21 to 27 Å in maximum length, were incorporated into bilayers made of phosphatidylcholine lipids with saturated acyl chains containing 14, 16, or 18 carbons, which are 36–50 Å in thickness. The effect of the peptide β-sheets on fluid- and gel-phase bilayers were studied with differential scanning calorimetry and circular dichroism spectroscopy. We show that AcWL₅ forms a stable, peptide-rich gel phase in all three lipids. The whole family of AcWL_n peptides appears to form similarly stable, nonmembrane-disrupting β-sheets in all bilayer phases and thicknesses. Bilayers containing up to 20 mol % peptide, which is the maximum concentration tested, formed gel phases with melting temperatures that were equal to, or slightly higher than, the pure lipid transitions. Given the range of peptide lengths and bilayer thicknesses tested, these experiments show that the AcWL_n family of membrane-inserted β-sheets exhibit remarkable structural plasticity in membranes.     

Detection of likely transmembrane β-strand regions in sequences of mitochondrial pore proteins using the Gibbs sampler

The mitochondrial channel VDAC is presumed to fold as a -barrel although the number and identity of transmembrane -strands in the protein are controversial. Previously, a novel multiple alignment algorithm called the Gibbs sampler was used to detect a residue-frequency motif in sequences of bacterial outer-membrane proteins that corresponds to transmembrane -strands in bacterial porins of known structure (Neuwaldet al., 1995,Protein Science,4, 1618. In the present study, this bacterial motif has been used to screen sets of mitochondrial membrane protein sequences, with matches occurring in only two classes of proteins: VDACs and the outer-membrane protein import pore (ISP42, MOM38). These results suggest a structural (and perhaps evolutionary) relatedness between the bacterial and mitochondrial pore proteins, with the mitochondrial subsequences that match the bacterial motif corresponding to transmembrane -strands as in the porins.     

Outer membrane β-barrel structure prediction through the lens of AlphaFold2

Most proteins found in the outer membrane of gram-negative bacteria share a common domain: the transmembrane β-barrel. These outer membrane β-barrels (OMBBs) occur in multiple sizes and different families with a wide range of functions evolved independently by amplification from a pool of homologous ancestral ββ-hairpins. This is part of the reason why predicting their three-dimensional (3D) structure, especially by homology modeling, is a major challenge. Recently, DeepMind's AlphaFold v2 (AF2) became the first structure prediction method to reach close-to-experimental atomic accuracy in CASP even for difficult targets. However, membrane proteins, especially OMBBs, were not abundant during their training, raising the question of how accurate the predictions are for these families. In this study, we assessed the performance of AF2 in the prediction of OMBBs and OMBB-like folds of various topologies using an in-house-developed tool for the analysis of OMBB 3D structures, and barrOs. In agreement with previous studies on other membrane protein classes, our results indicate that AF2 predicts transmembrane β-barrel structures at high accuracy independently of the use of templates, even for novel topologies absent from the training set. These results provide confidence on the models generated by AF2 and open the door to the structural elucidation of novel transmembrane β-barrel topologies identified in high-throughput OMBB annotation studies or designed de novo.