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. However, many questions remain regarding how the primary sequence of elastin (and the soluble precursor tropoelastin) governs the molecular structure, its organisation into a polymeric network, and the mechanical properties of the resulting material. The elasticity of elastin is known to be largely entropic in origin, a property that is understood to arise from both its disordered molecular structure and its hydrophobic character. Despite a high degree of hydrophobicity, elastin does not form compact, water-excluding domains and remains highly disordered. However, elastin contains both stable and labile secondary structure elements. Current models of elastin structure and function are drawn from data collected on tropoelastin and on elastin-like peptides (ELPs) but at the tissue level, elasticity is only achieved after polymerisation of the mature elastin. In tissues, the reticulation of tropoelastin chains in water defines the polymer elastin that bears elasticity. Similarly, ELPs require polymerisation to become elastic. There is considerable interest in elastin especially in the biomaterials and cosmetic fields where ELPs are widely used. This review aims to provide an up-to-date survey of/perspective on current knowledge about the interplay between elastin structure, solvation, and entropic elasticity.

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. At equilibrium, Cl- ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl- translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl- translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl- ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl- pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

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. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

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

Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium 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. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines.

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 ∼2.2 Šresolution. Overall structure and active sites of these enzymes are highly similar. In molecular dynamics (MD) calculations, only the defluorinating enzymes sample more compact conformations, which in turn allow more effective interactions with the small fluorine atom. Structural constraints, based on X-ray structures and MD calculations, correctly predict the defluorination activity of the homologous enzyme ST2570.

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

The heartbeat is initiated by voltage-gated sodium channel NaV1.5, 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. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.

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. One such probe covalently labeled CFTR at two sites as determined following trypsin digestion and analysis by tandem-mass spectrometry. One labeled peptide resides in the cytosolic loop 4 of CFTR and the other is located in tm8, proximal to the site identified by cryo-EM. Complementary data from functional and molecular dynamic simulation studies support a model, where VX-770 mediates potentiation via multiple sites in the CFTR protein.

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

Voltage-gated sodium (NaV) 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 NaV1.5 channels with IC50 = 11.4 nM. Here we reveal the structure of LqhIII bound to NaV1.5 at 3.3 Å resolution by cryo-EM. LqhIII anchors on top of voltage-sensing domain IV, wedged between the S1-S2 and S3-S4 linkers, which traps the gating charges of the S4 segment in a unique intermediate-activated state stabilized by four ion-pairs. This conformational change is propagated inward to weaken binding of the fast inactivation gate and favor opening the activation gate. However, these changes do not permit Na+ permeation, revealing why LqhIII slows inactivation of NaV channels but does not open them. Our results provide important insights into the structural basis for gating-modifier toxin binding, voltage-sensor trapping, and fast inactivation of NaV channels.

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. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient ß-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for ß-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

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 bc1 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 NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation.

Structural ordering of the Plasmodium berghei 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 P. falciparum (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. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to P. berghei CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.

Malaria is a significant health concern, killing about 400,000 people each year. While antimalarial drugs and insecticides have successfully reduced deaths over the last 20 years, the parasite that causes malaria is starting to gain resistance to these treatments. Vaccines offer an alternative route to preventing the disease. However, the most advanced vaccine currently available provides less than 50% protection. Vaccines work by encouraging the body to develop proteins called antibodies, which can recognize the parasite and trigger an immune response that blocks the infection. These antibodies target a molecule on the parasite's surface called circumsporozoite protein, or CSP for short. Therefore, having a better understanding of how antibodies interact with CSP could help researchers design more effective treatments. A lot of what is known about malaria has come from studying this disease in mice. However, it remained unclear whether antibodies produced in rodents combat the malaria-causing parasite in a similar manner to human antibodies. To answer this question, Kucharska, Thai et al. studied a mouse antibody called 3D11, which targets CSP on the surface of a parasite that causes malaria in rodents. The interaction between CSP and 3D11 was studied using three different techniques in order to better understand how the structure of CSP changes when bound by antibodies. The experiments showed that although CSP has a highly flexible structure, it forms a more stable, spiral-like architecture when bound to multiple copies of 3D11. A similar type of assembly was previously observed in studies investigating how CSP interacts with human antibodies. Further investigation revealed that the molecular connections between 3D11 and CSP share a lot of similarities with how human antibodies recognize CSP. These findings reveal how mammals evolved similar mechanisms for detecting and inhibiting malaria-causing parasites. This highlights the robust features antibodies need to launch an immune response against malaria, which could help develop a more effective vaccine.

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. In experimental studies, we use metal-ion bridges to show that promoting an M101-F99 bond directly activates Orai1, whereas disrupting this interaction triggers channel closure. Mutational analysis demonstrates that the methionine residue at this position has a unique combination of length, flexibility, and chemistry to act as an effective latch for the phenylalanine gate. Because sulfur-aromatic interactions provide additional stabilization compared to purely hydrophobic interactions, we infer that the six M101-F99 pairs in the hexameric channel provide a substantial energetic contribution to Orai1 activation.

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. In addition to this outer hydrophobic gate, several studies have postulated the presence of an inner gate formed by the basic residues R91, K87, and R83 in the inner pore. These positively charged residues were suggested to block ion conduction in closed channels via mechanisms involving either electrostatic repulsion or steric occlusion by a bound anion plug. However, in contrast to this model, here we find that neutralization of the basic residues dose-dependently abolishes both STIM1-mediated and STIM1-independent activation of Orai1 channels. Molecular dynamics simulations show that loss of the basic residues dehydrates the pore around the hydrophobic gate and stabilizes the pore in a closed configuration. Likewise, the severe combined immunodeficiency mutation, Orai1 R91W, closes the channel by dewetting the hydrophobic stretch of the pore and stabilizing F99 in a pore-facing configuration. Loss of STIM1-gating in R91W and in the other basic residue mutants is rescued by a V102A mutation, which restores pore hydration at the hydrophobic gate to repermit ion conduction. These results indicate that the inner pore basic residues facilitate opening of the principal outer hydrophobic gate through a long-range effect involving hydration of the outer pore.

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, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open-closed gating, but strikingly, at negative voltages where "normal" gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 µM Zn2+ Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

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 cholesterol-like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.

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. Empty protomer dynamics are also allosterically coupled to the bound protomer, driving conformational exchange at the active site and progress along the reaction coordinate. Here, we show that at high concentrations, a second substrate binds along the substrate-access channel of the occupied protomer, thereby dampening interprotomer dynamics and inhibiting catalysis. While a mutation (K152I) abrogates second site binding and removes inhibitory effects, it also precipitously lowers the maximum catalytic rate, implying a role for the allosteric pocket at low substrate concentrations, where only a single substrate engages the enzyme at one time. We show that this outer pocket first desolvates the substrate, whereupon it is deposited in the active site. Substrate binding to the active site then triggers the empty outer pocket to serve as an interprotomer allosteric conduit, enabling enhanced dynamics and sampling of activation states needed for catalysis. These allosteric networks and the ensuing changes resulting from second substrate binding are delineated using rigidity-based allosteric transmission theory and validated by nuclear magnetic resonance and functional studies. The results illustrate the role of dynamics along allosteric networks in facilitating function.

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. Recent studies have provided an atomistic description of the conformational ensemble of elastin-like proteins, and have begun to uncover how the interplay of local secondary structure, hydrophobicity and conformational disorder govern the structure, assembly and function of elastin. Monomeric elastin is a non-polar, glycine-rich, low-complexity, modular protein that remains predominantly disordered even in the crosslinked polymeric state, consistent with its function as an entropic elastomer. Unlike intracellular phase separation, which is reversible, phase separation of elastin and other matrix proteins proceeds to stabilization and clustering of condensed phase droplets and subsequent molecular organization into fibrillar, supramolecular structures. Short ß-sheets appear to mediate the interaction and organization of these phase-separated droplets and modulate the ultimate material properties of the matrix. Whether phase separation is intracellular or extracellular, reversible or network-forming, understanding the sequence determinants of such varied assembly behaviors and differential fates of the colloidal droplets will provide important insights into aberrant assembly with pathological consequences and elucidate fundamental principles for the rational design of biomimetic materials.

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 Ca2+ 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. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

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 weakness1,2. 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 Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis 5 owing to cation leak in both activated and inactivated states 6 . Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.

Mechanistic insights into allosteric regulation of the A2A 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, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.