The nicotinic acetylcholine receptor, a pentameric ligand-gated ion channel, converts the free energy of binding of the neurotransmitter acetylcholine into opening of its central pore. Here we ...present the first high-resolution structure of the receptor type found in muscle-endplate membrane and in the muscle-derived electric tissues of fish. The native receptor was purified from Torpedo electric tissue and functionally reconstituted in lipids optimal for cryo-electron microscopy. The receptor was stabilized in a closed state by the binding of α-bungarotoxin. The structure reveals the binding of a toxin molecule at each of two subunit interfaces in a manner that would block the binding of acetylcholine. It also reveals a closed gate in the ion-conducting pore, formed by hydrophobic amino acid side chains, located ∼60 Å from the toxin binding sites. The structure provides a framework for understanding gating in ligand-gated channels and how mutations in the acetylcholine receptor cause congenital myasthenic syndromes.
•High-resolution structure of a native muscle-type nicotinic acetylcholine receptor•Previously unresolved structural elements contribute to neurotoxin binding•Channel is stabilized in a closed conformation by α-bungarotoxin from snake venom•Transduction mechanism and myasthenic disease mutations
Rahman et al. report the high-resolution single-particle cryo-EM structure of a native muscle-type nicotinic acetylcholine receptor from the Torpedo electric ray, in complex with α-bungarotoxin from the banded krait. The structure was obtained in a lipidic environment shown to support channel function and reveals a closed, hydrophobic ion channel gate.
Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of ...three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.
Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and ...clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.
•AAGAB binds to the AP2 complex and is essential for clathrin-mediated endocytosis•AAGAB controls the sequential assembly of the AP2 adaptor complex•AAGAB stabilizes AP2 complex intermediates•A skin disease-causing mutation impairs the function of AAGAB
The AP2 complex is required for clathrin-mediated endocytosis. Gulbranson et al. identify AAGAB as a regulator of AP2 adaptor assembly, which guides the sequential association of AP2 subunits and stabilizes assembly intermediates. The function of AAGAB is disrupted by a mutation that causes punctate palmoplantar keratoderma type 1.
Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the ...negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their membrane integrity. However, many human pathogens, including Salmonella and Pseudomonas, have evolved lipid A modification mechanisms that result in resistance to CAMPs and related antibiotics such as Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N) to a phosphate group in lipid A is one such modification, frequently found in Pseudomonas isolated from cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate (PLP) dependent transaminase that catalyzes a crucial step in the pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present the 2.3 Å resolution crystal structure of an active site mutant of ArnB (K188A) in complex with the reaction intermediate aldimine formed by UDP-Ara4N and PLP. The sugar–nucleotide binding site is in a cleft between the subunits of the ArnB dimer with the uracil buried at the interface and the UDP ribose and phosphate groups exposed to the solvent. The Ara4N moiety is found in the 4C1 conformation and its positioning, stabilized by interactions with both the protein and cofactor, is compatible with catalysis. The structure suggests strategies for the development of specific inhibitors that may prove useful in the treatment of resistant bacteria such as Pseudomonas found in cystic fibrosis patients.
Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane ...fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.
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•The juxtamembrane motif of the v-SNARE is essential for vesicle fusion•The juxtamembrane motif directly destabilizes the lipid bilayer•The function of the juxtamembrane motif requires both polar and nonpolar residues•Linker insertions between SNARE and juxtamembrane motifs impair vesicle fusion
Membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, Rathore et al. demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE.
Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins ...required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H–H–D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid–base catalytic mechanism.
Zein was incorporated at 5% (w/w) to high amylose rice flours with different particle sizes (80–100, 100–140, and 140–250 μm) and the rice flour-zein composites were utilized to generate gluten-free ...noodles slit from sheeted doughs. Rice flours with smaller particle size showed higher starch damage whereas they had greater water hydration properties and higher pasting parameters. In addition, the fine particle size contributed to the elastic properties of the rice-zein composites. The ability of zein to generate a viscoelastic protein network at above its glass transition temperature, successfully produced gluten-free rice sheeted doughs regardless of particle size that could be slit into long and thin noodle strands without gluten. The rice-zein noodles with larger particle sizes had a rough surface while the noodle surface became smooth and flat at decreasing particle sizes. The noodle samples with rice flour having an intermediate particle size (100–140 μm) showed the highest values of maximum resistance and extensibility to extension that consequently contributed to the decreased cooking loss. This study provided a new opportunity for food manufacturers to improve the qualities of gluten-free rice noodles made from sheeted doughs by controlling particle size.
•Particle size effect of rice flour was investigated for gluten-free rice-zein noodles.•Composites with fine particles had higher pasting profile (closely-packed structure).•Composites formed a viscoelastic network, generating sheeted dough and noodle strand.•SEM analysis showed smooth and flat surface of composite noodle with fine particle.•Noodle (100–140 μm particle) had greater resistance to extension/lower cooking loss.
This study aimed to explore the feasibility of zein as a gluten alternative in preparing sheeted doughs with rice flours containing different amylose contents (12, 19, and 26%) that were successfully ...slit into noodle strands. Rice-zein mixtures with lower amylose contents exhibited lower pasting parameters and the high amylose paste samples possessed more elastic properties. Higher water absorption was observed in the low amylose rice-zein mixture whereas the use of zein were effective in increasing the stability of rice dough regardless of amylose content. In case of thermal conductivity, the noodle doughs prepared with high amylose rice-zein mixture had low thermal conductivity, probably implying a long cooking time. The structure of the rice-zein noodles had a tendency to become firmer with increasing levels of amylose by showing greater breaking stress and resistance to extension that could be correlated to reduced cooking loss.
•Zein was used as a gluten alternative in rice noodles with different amylose contents.•Mixing rice flour with zein above Tg of zein successfully generated rice dough sheets.•High amylose rice (HAR)-zein pastes exhibited greater pasting and elastic properties.•Low thermal conductivity was observed in HAR-zein noodle dough (long cooking time).•HAR-zein noodles showed a firm texture that contributed to their reduced cooking loss.
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