Structural and physical basis for the elasticity of elastin.

Elastin function is to endow vertebrate tissues with elasticity so that they can adapt to local mechanical constraints. The hydrophobicity and insolubility of the mature elastin polymer have hampered studies of its molecular organisation and structure-elasticity relationships. Nevertheless, a growing number of studies from a broad range of disciplines have provided invaluable insights, and several structural models of elastin have been proposed.

Molecular dynamics study of Cl permeation through cystic fibrosis transmembrane conductance regulator (CFTR).

The recent elucidation of atomistic structures of Cl channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein.

Structural basis of inhibition by antibodies binding to the circumsporozoite protein repeats.

Malaria is a global health burden, with (Pf) and (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP.

Defluorination Capability of l-2-Haloacid Dehalogenases in the HAD-Like Hydrolase Superfamily Correlates with Active Site Compactness.

l-2-Haloacid dehalogenases, industrially and environmentally important enzymes that catalyse cleavage of the carbon-halogen bond in S-2-halocarboxylic acids, were known to hydrolyse chlorinated, brominated and iodinated substrates but no activity towards fluorinated compounds had been reported. A screen for novel dehalogenase activities revealed four l-2-haloacid dehalogenases capable of defluorination. We now report crystal structures for two of these enzymes, Bpro0530 and Rha0230, as well as for the related proteins PA0810 and RSc1362, which hydrolyse chloroacetate but not fluoroacetate, all at ∼2.

Open-state structure and pore gating mechanism of the cardiac sodium channel.

The heartbeat is initiated by voltage-gated sodium channel Na1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure.

Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator.

Ivacaftor (VX-770) was the first cystic fibrosis transmembrane conductance regulator (CFTR) modulatory drug approved for the treatment of patients with cystic fibrosis. Electron cryomicroscopy (cryo-EM) studies of detergent-solubilized CFTR indicated that VX-770 bound to a site at the interface between solvent and a hinge region in the CFTR protein conferred by transmembrane (tm) helices: tm4, tm5, and tm8. We re-evaluated VX-770 binding to CFTR in biological membranes using photoactivatable VX-770 probes.

Structural basis for voltage-sensor trapping of the cardiac sodium channel by a deathstalker scorpion toxin.

Voltage-gated sodium (Na) channels initiate action potentials in excitable cells, and their function is altered by potent gating-modifier toxins. The α-toxin LqhIII from the deathstalker scorpion inhibits fast inactivation of cardiac Na1.5 channels with IC = 11.

The evolutionary background and functional consequences of the rs2071307 polymorphism in human tropoelastin.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens.

Structural ordering of the circumsporozoite protein repeats by inhibitory antibody 3D11.

Plasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 - the leading malaria vaccine against (Pf) - has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear.

A sulfur-aromatic gate latch is essential for opening of the Orai1 channel pore.

Sulfur-aromatic interactions occur in the majority of protein structures, yet little is known about their functional roles in ion channels. Here, we describe a novel molecular motif, the M101 gate latch, which is essential for gating of human Orai1 channels via its sulfur-aromatic interactions with the F99 hydrophobic gate. Molecular dynamics simulations of different Orai variants reveal that the gate latch is mostly engaged in open but not closed channels.

NMR Structure and Dynamics Studies of Yeast Respiratory Supercomplex Factor 2.

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble α helices and five putative transmembrane (TM) α helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation.

The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate.

Store-operated Orai1 channels regulate a wide range of cellular functions from gene expression to cell proliferation. Previous studies have shown that gating of Orai1 channels is regulated by the outer pore residues V102 and F99, which together function as a hydrophobic gate to block ion conduction in resting channels. Opening of this gate occurs through a conformational change that moves F99 away from the permeation pathway, leading to pore hydration and ion conduction.

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels.

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this "gating pore" when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hH1, but proton current flows through the same pore in open channels.

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) is involved in lysosomal cholesterol export.

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterollike inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes.

Substrate-Based Allosteric Regulation of a Homodimeric Enzyme.

Many enzymes operate through half-of-the sites reactivity wherein a single protomer is catalytically engaged at one time. In the case of the homodimeric enzyme, fluoroacetate dehalogenase, substrate binding triggers closing of a regulatory cap domain in the empty protomer, preventing substrate access to the remaining active site. However, the empty protomer serves a critical role by acquiring more disorder upon substrate binding, thereby entropically favoring the forward reaction.

Role of Liquid-Liquid Phase Separation in Assembly of Elastin and Other Extracellular Matrix Proteins.

Liquid-liquid phase separation resulting in formation of colloidal droplets has recently attracted attention as a mechanism for rapid and transient assembly of intracellular macromolecules into functional units. Phase separation also appears to be a widespread and evolutionarily ancient mechanism for organization of proteins of the extracellular matrix into fibrillar, polymeric assemblies. Elastin, which provides the physical properties of extensibility and elastic recoil to large arteries, lungs and other tissues, is the best-characterized extracellular matrix protein whose polymeric assembly is initiated by phase separation.

Structural basis for gating pore current in periodic paralysis.

Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Na1.4 or the calcium channel Ca1.

Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating.

Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1-4 whose perturbation activates Orai1 channels independently of STIM1.

Mechanistic insights into allosteric regulation of the A adenosine G protein-coupled receptor by physiological cations.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, F NMR is used to delineate the effects of cations on functional states of the adenosine A GPCR. While Na reinforces an inactive ensemble and a partial-agonist stabilized state, Ca and Mg shift the equilibrium toward active states.

Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation.

The respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a "charge zipper" motif.

Molecular recognition and packing frustration in a helical protein.

Biomolecular recognition entails attractive forces for the functional native states and discrimination against potential nonnative interactions that favor alternate stable configurations. The challenge posed by the competition of nonnative stabilization against native-centric forces is conceptualized as frustration. Experiment indicates that frustration is often minimal in evolved biological systems although nonnative possibilities are intuitively abundant.

The liquid structure of elastin.

The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are hydrophobic, disordered, and undergo liquid-liquid phase separation upon self-assembly. Despite extensive study, the structure of elastin remains controversial.

Orkambi® and amplifier co-therapy improves function from a rare mutation in gene-edited cells and patient tissue.

The combination therapy of lumacaftor and ivacaftor (Orkambi) is approved for patients bearing the major cystic fibrosis (CF) mutation: It has been predicted that Orkambi could treat patients with rarer mutations of similar "theratype"; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function-similar to ΔF508-CFTR, are unlikely to yield a robust Orkambi response.

Somatostatin binds to the human amyloid β peptide and favors the formation of distinct oligomers.

The amyloid β peptide (Aβ) is a key player in the etiology of Alzheimer disease (AD), yet a systematic investigation of its molecular interactions has not been reported. Here we identified by quantitative mass spectrometry proteins in human brain extract that bind to oligomeric Aβ1-42 (oAβ1-42) and/or monomeric Aβ1-42 (mAβ1-42) baits. Remarkably, the cyclic neuroendocrine peptide somatostatin-14 (SST14) was observed to be the most selectively enriched oAβ1-42 binder.