Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.     

Dimensions of the ion channel in neuronal nicotinic acetylcholine receptor as estimated from analysis of conformation-activity relationships of open-channel blocking drugs

Summary Relationship between the size of the molecule in the series of organic ions Et₃N–(CH₂)₅–N⁺R¹R²R³ (R ⁱ -alkyl or cycloalkyl substituents) and their abilities to block nicotinic acetylcholine receptors (AChRs) due to their open-channel blockade in the neurons of autonomic ganglia and in frog end-plate was analyzed.All low-energy equilibrium conformations of the drugs were calculated by the molecular mechanics method. A unique rectangular channel profile 6.1×8.3 Å. for which the best correlation between blocking activity of the drugs and total population of their conformations being able to penetrate into the channel, was deduced from all those tested.     

Acetylcholine receptor binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with K_D approx. 10^?8. The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.     

Voltage-dependent cationic channel of Escherichia coli

Verapamil and dimethylcurine are Ca²⁺ entry blockers of essentially different chemical structures which presumably bind to the same arylalkylamine receptor of the L-type Ca channel. A systematic conformational analysis of methoxyverapamil (D-600) and dimethylcurine has been carried out using a molecular mechanics method. The lowest minimum-energy conformations of D-600 are predisposed to chelate Ca²⁺ by four oxygen atoms of the stacked methoxyphenyl moieties. Comparison of the lowest energy conformations of D-600-Ca²⁺ and dimethylcurine revealed a similar spatial disposition of cationic groups and methoxyphenyl moieties in the two compounds. A three-dimensional model of arylalkylamine receptor was suggested which incorporates two nucleophilic areas of the Ca channel. Dimethylcurine binds to these areas by its quaternary amine functions, whereas D-600 does so by amine function and via coordinated Ca²⁺. The results support the hypotheses on ternary complex formation between the ligands of Ca channel, their receptors, and Ca²⁺.     

Characterization of quercetin binding site on DNA gyrase

Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs. Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage. It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA. We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a K(D) value of 15 microM and inhibits ATPase activity of gyrase B. Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin. The structural model of quercetin-gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck. We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase.     

Inhibition of estradiol binding to its receptor by the cupric ion

 Treatment of human recombinant estrogen receptor (hER) expressed in yeast with very low concentrations of the cupric ion decreased its ability to bind [³H]estradiol ([³H]E₂) and [¹²⁵I]tamoxifen aziridine (minimal Cu²⁺ concentration: 1 nM). This decrease was reflected in a loss of immunoreactivity for monoclonal antibodies raised against the hormone binding domain (HBD). An ER recombinant expressing solely the HBD confirmed that the ion operated at this level. Cysteines located within this domain contributed to the inhibitory action of the Cu²⁺ in view of a partial restoration of the [³H]E₂ binding activity with β-mercaptoethanol. Histidines were also implicated since the influence of Cu²⁺ on the [³H]E₂ binding parameters (Scatchard plot analysis) was maintained after oxidation of thiol groups by methyl methanethiosulfonate, and partly reversed by imidazole. Received: 10 December 1997 / Accepted: 26 June 1998     

A molecular model for the bilayer helices of the acetylcholine receptor including an acetylcholine binding site

A molecular model for the bilayer helices of the acetylcholine receptor is constructed from the 7 channel elements and the 17 hydrophobic helices of the 5 protein subunits. The acetylcholine binding site and the opening to the ion channel are included.     

Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics

The Ω-loop at the active site of β-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of β-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered β-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the Ω-loop and to detect the binding of diverse substrates. Our results show that this engineered β-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the Ω-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the Ω-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum β-lactam antibiotics. The oxyimino-cephalosporin-specific fluorescence profile of our engineered β-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.     

Acetylcholine synthesis in human CSF: Implications for study of central cholinergic metabolism

Investigation of neurological diseases involving central cholinergic dysfunction has led to numerous studies seeking a peripheral marker of cholinergic activity in brain. The main objective of these studies was to determine whether the ACh synthesizing activity present in human CSF was due to the presence of the enzyme choline acetyltransferase (ChAT; 68kDa). When CSF was fractionated into low and high molecular weight (M_r) components, 80% of the ACh synthesizing activity (AChSA) was found to be associated with the fraction <10 kDa. The remaining 20% was evenly distributed among fractions in the 5–30, 30–50, 50–300, and 300 kDa fractions. Although boiling destroyed all activity >10 kDa, the ChAT inhibitor NVP, at concentrations equal to or greater than that required to inhibit ChAT in human cortical tissue, did not alter the ACh-SA in either fraction. Results indicate that normal human CSF does not contain ChAT and all ACh-SA in CSF reflects non-enzymatic imidazole/histidine-like catalyzed synthesis.     

The low-affinity lipid binding site of the non-specific lipid transfer protein. Implications for its mode of action.

The non-specific lipid transfer protein (nsL-TP) from bovine liver was studied by using the following fluorescent lipid analogs: phosphatidylcholine species with a sn-2-pyrenylacyl-chain of different length [Pyr(x)PC], sn-2-pyrenyldecanoyl-labelled phosphatidylinositol [Pyr(10)PI], -phosphatidylinositol 4-phosphate [Pyr(10)PIP], -phosphatidylinositol 4,5-bisphosphate [Pyr(10)PIP2] and dehydroergosterol. These analogs provided information on the effect of hydrophobicity and charge on lipid binding and transfer by nsL-TP. Binding of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length. Under equilibrium conditions, the fraction of nsL-TP that carried a PC molecule did not exceed 8%, which is consistent with a low affinity binding site. Also nsL-TP-mediated transfer of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length and was highly correlated with spontaneous transfer. Binding of the phosphoinositides increased in the order Pyr(10)PI less than Pyr(10)PIP less than Pyr(10)PIP2, indicating that an increase in lipid negative charge stimulates binding. The transfer of the phosphoinositides, however, decreased in the same order, which suggests that a high negative charge impairs the dissociation of the phospholipid from nsL-TP. Cholesterol, at concentrations up to 50 mol% in the donor membrane, hardly affected binding and transfer of Pyr(6)PC, strongly suggesting that nsL-TP has no high binding affinity for cholesterol. In agreement with this, binding of dehydroergosterol to nsL-TP was not detectable. Despite this apparently negligible affinity, nsL-TP-mediated transfer of dehydroergosterol was in the same order as that of Pyr(6)PC. The results are interpreted to indicate that transfer of lipids by nsL-TP involves the formation of a putative low-affinity lipid-protein complex. This formation is enhanced when lipid hydrophobicity decreases or lipid negative charge increases. Based on the binding and transfer data, the mode of action of nsL-TP is discussed in terms of change in free energy.     

IRBIT: A regulator of ion channels and ion transporters

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca^2 + channel inositol 1,4,5-trisphosphate (IP₃) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na⁺/HCO₃⁻ co-transporter NBCe1-B, the Na⁺/H⁺ exchanger NHE3, the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl⁻/HCO₃⁻ exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.     

Sequence analysis,structure and binding site prediction of Sigma 1 receptor protein by in silico method

Sigma 1 Receptor is a subtype of opioid receptor that participates in membrane remodeling and cellular differentiation in thenervous system. Sigma1 Receptor protein with amino acid length ranging from 229 is widely distributed in the liver andmoderately in the intestine, kidney, white pulp of the spleen, adrenal gland, brain, placenta and the lung. In this study, the threedimensional structure for sigma 1 receptor protein has been developed by in- silico analysis based on evolutionary trace analysis of37 sigma proteins from different sources. The present work focus on identification of functionally important residues and itsinteraction with antipsychotic drugs reported in literature.     

Evidence for an extended hydrogen bond network in the binding site of the nicotinic receptor: role of the vicinal disulfide of the alpha1 subunit

The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a β turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the β turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.     

Order changes within receptor systems upon ligand binding: receptor tightening/oligomerisation and the interpretation of binding parameters

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of "thermodynamic discrimination" between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the DeltaH and DeltaS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.     

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.     

Insect nicotinic acetylcholine receptor: conserved neonicotinoid specificity of [(3)H]imidacloprid binding site

The insect nicotinic acetylcholine receptor (nAChR) is a major target for insecticide action. The rapidly expanding use of neonicotinoid insecticides of varied structures makes it increasingly important to define similarities and differences in their action, particularly for the first-generation chloropyridinyl compounds versus the second-generation chlorothiazolyl derivatives. We have shown with Musca domestica that a convenient and relevant determination of the neonicotinoid insecticide target is a binding site assay with [(3)H]imidacloprid ([(3)H]IMI). This study uses membranes from the aphids MYZUS: persicae and Aphis craccivora and from heads of the flies DROSOPHILA: melanogaster and Musca domestica to characterize the [(3)H]IMI binding sites relative to their number and possible species variation in structure-activity relationships. With emphasis on commercial neonicotinoids, six potent chloropyridinyl compounds are compared with the corresponding six chlorothiazolyl analogues (syntheses are given for chemicals prepared differently than previously described). The preference for chloropyridinyl versus chlorothiazolyl is not dependent on the insect species examined but instead on other structural features of the molecule. The chlorothiazolyl substituent generally confers higher potency in the clothianidin and desmethylthiamethoxam series and the chloropyridinyl moiety in the imidacloprid, thiacloprid, acetamiprid, and nitenpyram series. Two chlorothiazolyl compounds compete directly with the chloropyridinyl [(3)H]IMI for the same binding sites in MYZUS: and DROSOPHILA: membranes. This study shows conserved neonicotinoid specificity of the [(3)H]IMI binding site in each of the four insect species examined.     

Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes

Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.