Structural basis of ligand recognition in 5-HT3 receptors

The 5‐HT₃ receptor is a pentameric serotonin‐gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti‐emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5‐HT₃ receptor. In the serotonin‐bound structure, we observe hydrophilic interactions with loop E‐binding site residues, which might enable transitions to channel opening. In the granisetron‐bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5‐HT₃ receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high‐affinity ligand binding in the human 5‐HT₃ receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti‐emetics in the 5‐HT₃ receptor.     

Detailed conformational dynamics of juxtamembrane region and activation loop in c-Kit kinase activation process

The stem cell factor receptor (c-Kit) plays critical roles in initiating cell growth and proliferation. Its kinase functional abnormality has been thought to associate with several human cancers. The regulation of c-Kit kinase activity is achieved by phosphorylation on the residues Tyr568 and Tyr570 within juxtamembrane region (JMR) and subsequent structural transition of JMR and activation loop (A-loop). However, the detailed conformational dynamics of JMR and A-loop are far from clear, especially whether their conformational changes are coupled or not during the kinase activation transition. In this investigation, the complete conformational transition pathway was determined using a series of nanosecond conventional molecular dynamics (MD) and targeted molecular dynamics (TMD) simulations in explicit water systems. The results of the MD simulations show that the phosphorylation of residues Tyr568 and Tyr570 within JMR induces the detachment of JMR from the kinase C-lobe and increases the fluctuation in the structure of JMR, thus appearing to initiate the kinase activation process. During the course of the TMD simulation, which characterizes the conformational transition of c-Kit from autoinhibitory to activated state, the JMR undergoes a rapid departure from the allosteric binding site and drifts into solvent, followed by the conformational flip of A-loop from inactive (fold) state to active (extended) state. A change in the orientation of helix alphaC in response to the motion of JMR and A-loop has also been observed. The computational results presented here indicate that the dissociation of JMR from the kinase domain is prerequisite to c-Kit activation, which is consistent with previous experiments.     

A Single Conserved Leucine Residue on the First Intracellular Loop Regulates ER Export of G Protein‐Coupled Receptors

The intrinsic structural determinants for export trafficking of G protein‐coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The α_2B‐adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates α_2B‐AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of α_2B‐AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of β₂‐AR, α_1B‐AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.     

Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Common Misconceptions about Recycling

The recycling of material resources lies at the heart of the industrial ecology (IE) metaphor. The very notion of the industrial ecosystem is motivated by the idea that we should learn from natural ecosystems how to “close the loop.” Recycling is not just central to IE, it is part of everyday life. Unfortunately, how the IE community and the public at large think about recycling includes several misconceptions that have the potential to misguide environmental assessments, policies, and actions that deal with recycling and thus undermine its environmental potential. One misconception stems from naïve assumptions regarding recycled material displacing primary production. Two others assert the environmental advantages of recycling material multiple times, or at least in a closed loop. A final misconception is the assumption that the distinction between closed and open recycling loops is generally useful. This article explains why these misconceptions are flawed, discusses the implications, and presents an alternative set of principles to better harness the potential environmental benefits of closing material loops.     

Cytoplasmic regions adjacent to the M3 and M4 transmembrane segments influence expression and function of alpha7 nicotinic acetylcholine receptors. A study with single amino acid mutants

We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of alpha7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of alpha-bungarotoxin binding sites and acetylcholine-evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.     

The innate immune system in the intestine

The innate immune system provides the first line of host defense against invading pathogens. Innate immune responses are initiated by germline-encoded PRR, which recognize specific structures expressed by microorganisms. TLR are a family of PRR which sense a wide range of microorganisms, including bacteria, fungi, protozoa and viruses. TLR are also expressed in the intestine and are critical for intestinal homeostasis. Recently, cytoplasmic PRR, such as NLR and RLR, have been shown to detect pathogens that have invaded the cytosol. One of the NLR, NOD2, is thought to be involved in the pathogenesis of Crohn's disease. This review focuses on the innate immune responses triggered by PRR in the intestine.     

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights into agonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.     

A triple arg motif mediates α(2B)-adrenergic receptor interaction with Sec24C/D and export

Recent studies have demonstrated that cargo exit from the endoplasmic reticulum (ER) may be directed by ER export motifs recognized by components of the coat protein II (COPII) vesicles. However, little is known about ER export motifs and vesicle targeting of the G protein-coupled receptor (GPCR) superfamily. Here, we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α(2B)-adrenergic receptor (α(2B)-AR). The 3R motif mediates α(2B)-AR interaction with Sec24C/D and modulates ER exit, cell surface transport and function of α(2B)-AR. Furthermore, export function of the 3R motif is independent of its position within α(2B)-AR and can be conferred to CD8 glycoprotein. These data provide the first evidence implicating that export of GPCRs is controlled by code-directed interactions with selective components of the COPII transport machinery.     

Intracellular third loop-C-terminal tail interaction in prostaglandin EP3beta receptor

The secondary structures of and the interactions between the intracellular domains (the three loops and the C-terminal tail) of the mouse-derived prostaglandin E2 receptor EP3β subtype were investigated using peptides mimicking the domains. The N-termini of the peptides were palmitoylated to anchor on unilamellar vesicles composed of phosphatidylserine, enriched in the cytoplasmic leaflet of mammalian plasma membranes. Circular dichroism spectroscopy revealed that the peptides corresponding to the intracellular third loop (i3) and the C-terminus (C-term) assumed β-sheet and associated α-helical structures, respectively. A structural change was observed when i3 was mixed with C-term, indicating an interaction between them. Fluorescence experiments showed that i3 suppressed the self-association of C-term, confirming the interaction. These results demonstrate for the first time specific interaction between the intracellular third loop and the C-terminus. A model is proposed for the activation of the receptor.     

Amino‐acid composition after loop deletion drives domain swapping

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain‐swapped oligomer over the monomer. While it is known that the deletion of loops between β‐strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop‐deletion mutants of the non‐domain‐swapping protein monellin, frame‐shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping.     

Functional analysis of the distal region of the third intracellular loop of PROKR2

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270–272) resulted in undetectable cell surface expression, whereas RKR (264–266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.     

Axonal targeting of the 5-HT1B serotonin receptor relies on structure-specific constitutive activation

By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G-protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and spontaneous endocytosis for the axonal 5-HT(1B) R, as compared to the somatodendritic 5-HT(1A) R, both in non-neuronal cells and neurons. Activation-dependent constitutive endocytosis is crucial for axonal targeting, because inverse-agonist treatment, which prevents constitutive activation, leads to atypical accumulation of newly synthesized 5-HT(1B) Rs on the somatodendritic plasma membrane. Using receptor chimeras composed of different domains from 5-HT(1A) R and 5-HT(1B) R, we show that the complete third intracellular loop of 5-HT(1B) R is necessary and sufficient for constitutive activation and efficient axonal targeting, both sensitive to inverse-agonist treatment. These results suggest that activation and targeting of 5-HT(1B) Rs are intimately interconnected in neurons.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.     

Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor

Deuterium oxide solutions of a triple-helical polysaccharide schizophyllan, undergoing an order-disorder transition centered around 17 degrees C, were studied by the time-domain reflectometry (TDR) to obtain dielectric dispersions in the solution and frozen states. In the solution state, the dispersion below the transition temperature is resolved in three dispersions (relaxation times at 0 degrees C) ascribed to side chain glucose residue (1; 102 ns), structured water (s; 2.0 ns) and bulk water (h), respectively, from low to high frequencies. Bulk water is divided into slow water (h2; 0.04 ns) and free or pure water (h1; 0.02 ns). Above the transition temperature structured water almost disappears and is compensated by slow water. Structured water is similar to bound water for proteins but different from it because of this transition behavior. Another dispersion (l) seen at the lowest frequency is assigned to the rotation of side-chain glucose residue coupled with hydrated water. Parts of this dispersion and structured water are suggested to constitute bound water. In the frozen state were observed a major dispersion (h; 0.14 ns) and a minor one (m; 28 ns), which were ascribed to considerably mobile and less mobile waters. They are similar to but not exactly the same as that for unfreezable water in bovine serum albumin solutions argued by Miura et al. (Biopolymers, 1995, Vol. 36, p. 9). Water is molded into different structures by the triple helix.