Signalling assemblies: the odds of symmetry.

The assembly of proteins into complexes is fundamental to nearly all biological signalling processes. Symmetry is a dominant feature of the structures of experimentally determined protein complexes, observed in the vast majority of homomers and many heteromers. However, some asymmetric structures exist, and asymmetry also often forms transiently, intractable to traditional structure determination methods. Here, we explore the role of protein complex symmetry and asymmetry in cellular signalling, focusing on receptors, transcription factors and transmembrane channels, among other signalling assemblies. We highlight a recurrent tendency for asymmetry to be crucial for signalling function, often being associated with activated states. We conclude with a discussion of how consideration of protein complex symmetry and asymmetry has significant potential implications and applications for pharmacology and human disease.

Asymmetric perturbations of signalling oligomers.

This review focuses on rapid and reversible noncovalent interactions for symmetric oligomers of signalling proteins. Symmetry mismatch, transient symmetry breaking and asymmetric perturbations via chemical (ligand binding) and physical (electric or mechanic) effects can initiate the signalling events. Advanced biophysical methods can reveal not only structural symmetries of stable membrane-bound signalling proteins but also asymmetric functional transition states. Relevant techniques amenable to distinguish between symmetric and asymmetric architectures are discussed including those with the capability of capturing low-populated transient conformational states. Typical examples of signalling proteins are overviewed for symmetry breaking in dimers (GPCRs, growth factor receptors, transcription factors); trimers (acid-sensing ion channels); tetramers (voltage-gated cation channels, ionotropic glutamate receptor, CNG and CHN channels); pentameric ligand-gated and mechanosensitive channels; higher order oligomers (gap junction channel, chaperonins, proteasome, virus capsid); as well as primary and secondary transporters. In conclusion, asymmetric perturbations seem to play important functional roles in a broad range of communicating networks.

Allostery in pharmacology: thermodynamics, evolution and design.

This review focuses on basic models of allostery, the ambiguous application of the allosteric term in pharmacology illustrated by receptors, the role of thermodynamics in allosteric mechanisms, evolution and design of allostery. The initial step of ligand activation is closure of the agonist-binding cavity. Large entropy increases accompany the agonist-elicited conformational changes of pentameric ligand-gated ion channels due to cavity closure and rearrangement of transmembrane helices. The effects of point mutations on thermodynamic parameters of binding and function can reveal energetic coupling of neighbouring (and distant) amino acid residues in activation. High-order double-mutant cycle analysis and rate-equilibrium linear free-energy relationships can identify the trajectory and conformational spread of activation. Protein assembly and allostery can be deduced from colocalization and physicochemical principles. Molecular evolution has led from homooligomerization of protomers to heterotropic cooperativity and to allosteric regulation. Examples are discussed such as similar paths of protein (dis)assembly and evolution, irreversible evolution, statistical analysis of sequence homology revealing coevolution, different impacts of adaptation and evolution on hemoglobin, and the flagellar motor switch of bacteria. The driving force of dynamic allostery is associated with funnel-like free energy landscapes of protein binding and shifts in conformational fluctuations upon binding. Allostery can be designed based on our increasing knowledge of natural allosteric mechanisms and evolution. The allosteric principle has been applied for various bio/macro/molecular and signal transduction systems as well as in cognitive sciences.

Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function.

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA(A)) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [(3)H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3µM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [(3)H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC(50)=150nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA(A) receptors containing α6 and δ subunits. In contrast, [(3)H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC(50) ~80nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [(3)H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [(3)H]EBOB binding with EC(50) ~0.5µM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1µM GABA in cerebellar granule cells, for 5α-THDOC with EC(50,1) ~16nM and EC(50,2) ~1.3µM. Differences in peak current enhancements in the absence minus presence of 0.1mM furosemide corresponding to α6ßδ GABA(A) receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA(A) receptor populations.

Ligand-gated pentameric ion channels, from binding to gating.

X-ray structures of molluscan acetylcholine-binding proteins and procaryotic proton-activated ion channels (ELIC and GLIC) enable us to model the ligand binding and activation mechanism of ligand-gated pentameric ion channels. Common versus distinct features can be deduced from the binding of agonists, antagonists and allosteric modulators in subunit interfaces of nicotinic acetylcholine, A-type gamma-aminobutyric acid, glycine and 5-HT(3)-type serotonin receptors. Ligand interactions in subunit interfaces elicit conformational waves from the closure of the agonist-binding cavity through binding loops, beta-strands and transmembrane helices to pore gating.

Synthesis of heteroaromatic tropeines and heterogeneous binding to glycine receptors.

Heteroaromatic carboxylic esters of (nor)tropine were synthesized. Tropine esters displaced [(3)H]strychnine binding to glycine receptors of rat spinal cord with low Hill slopes. Two-site displacement resulted in nanomolar IC(50,1) and micromolar IC(50,2) values, and IC(50,2)/IC(50,1) ratios up to 615 depending on the heteroaromatic rings and N-methyl substitution. Nortropeines displayed high affinity and low heterogeneity. IC(50,1) and IC(50,2) values of tropeines did not correlate suggesting different binding modes/sites. Glycine potentiated only the nanomolar displacement reflecting positive allosteric interactions and potentiation of ionophore function. Affinities of three (nor)tropeines were different for glycine receptors but identical for 5-HT(3) receptors.

Different binding modes of tropeines mediating inhibition and potentiation of alpha1 glycine receptors.

Tropeines are bidirectional modulators of native and recombinant glycine receptors (GlyRs) and promising leads for the development of novel modulatory agents. Tropisetron potentiates and inhibits agonist-triggered GlyR currents at femto- to nanomolar and micromolar concentrations respectively. Here, the potentiating and inhibitory effects of another tropeine, 3alpha-(3'-methoxy-benzoyloxy)nortropane (MBN) were examined by voltage-clamp electrophysiology at wild type and mutant alpha1 GlyRs expressed in Xenopus laevis oocytes. Several substitutions around the agonist-binding cavity of the alpha1 subunit interface (N46C, F63A, N102A, R119K, R131A, E157C, K200A, Y202L and F207A) were found to reduce or eliminate MBN inhibition of glycine activation. In contrast, the binding site mutations Q67A, R119A and S129A which did not affect MBN inhibition abolished the potentiation of chloride currents elicited by low concentrations of the partial agonist taurine following pre-incubation with MBN. Thus, potentiation and inhibition involve distinct binding modes of MBN in the inter-subunit agonist-binding pocket of alpha1 GlyRs. Homology modelling and molecular dynamics simulations disclosed two distinct docking modes for MBN, which are consistent with the differential effects of individual binding site substitutions on MBN inhibition and potentiation respectively. Together these results suggest that distinct binding modes at adjacent binding sites located within the agonist-binding pocket of the GlyR mediate the bidirectional modulatory effects of tropeines.

Synthesis and receptor binding of new thieno[2,3-d]-pyrimidines as selective ligands of 5-HT(3) receptors.

With the aim to develop new potent and selective ligands of 5-HT(3)-type serotonin receptors and to acquire more information on their structure-affinity relationships, new thieno[2,3-d]pyrimidine derivatives 32-39 were synthesized and their binding to 5-HT(3) versus 5-HT(4 )receptors was studied. Some of these new compounds exhibit good affinity for cortical 5-HT(3) receptors, but not for 5-HT(4) receptors. Among these derivatives, 6-ethyl-4-(4-methyl-1-piperazinyl)-2-(methylthio)thieno[2,3-d]pyrimidine 32 is the most potent ligand (K(i) = 67 nM); it behaves as a competitive antagonist of the 5-HT(3) receptor function in the guinea pig colon. Its binding interactions with 5-HT(3A )receptors were analysed by using receptor modelling and comparative docking.

Synthesis of (nor)tropeine (di)esters and allosteric modulation of glycine receptor binding.

(Hetero)aromatic mono- and diesters of tropine and nortropine were prepared. Modulation of [3H]strychnine binding to glycine receptors of rat spinal cord was examined with a ternary allosteric model. The esters displaced [3H]strychnine binding with nano- or micromolar potencies and strong negative cooperativity. Coplanarity and distance of the ester moieties of diesters affected the binding affinity being nanomolar for isophthaloyl-bistropane and nortropeines. Nortropisetron had the highest affinity (K(A) approximately 10 nM). Two esters displayed negative cooperativity with glycine in displacement, while three esters of low-affinity and nortropisetron exerted positive cooperativity with glycine.

Hyperekplexia mutation R271L of alpha1 glycine receptors potentiates allosteric interactions of nortropeines, propofol and glycine with [3H]strychnine binding.

Human alpha1 and hyperekplexia mutant alpha1(R271L) glycine receptors (GlyRs) were transiently expressed in human embryonic kidney 293 cells for [3H]strychnine binding. Binding parameters were determined using a ternary allosteric model. The hyperekplexia mutation increased the positive cooperativity of 0.3 mM propofol and glycine binding by about six times: the cooperativity factor beta was 0.26 for alpha1 GlyRs and 0.04 for alpha1(R271L) GlyRs. Thus, propofol restored the potency of glycine impaired by the mutation. Five nortropeines, i.e. substituted benzoates of nortropine and a new compound, nortropisetron were prepared and also examined on [3H]strychnine binding. They showed nanomolar displacing potencies amplified by the hyperekplexia mutation. The affinity of nor-O-zatosetron (2.6 nM) is one of the highest reported for GlyRs. This binding test offers an in vitro method to evaluate agents against neurological disorders associated with inherited mutations of GlyRs.

Allosteric modulation of [3H]EBOB binding to GABAA receptors by diflunisal analogues.

Allosteric modulatory effects of 12 biphenyl derivatives of diflunisal and two fenamates were studied on A-type receptors of GABA (GABAAR) via [3H]4'-ethynylbicycloorthobenzoate (EBOB) binding to synaptic membrane preparations of rat forebrain. A simplified ternary allosteric model was used to determine binding affinities of the compounds and the extents of cooperativity with GABA. Structure activity analysis revealed that 4-hydroxy substituents of the biphenyls contribute to their micromolar binding affinities more than 3-carboxyl groups. Electron-withdrawing fluorinated substituents, especially in ortho position, were also advantageous. These factors also strongly enhanced the cooperativity with GABA binding. The correlation between displacing potency of the allosteric agents and cooperativity with GABA suggests that these processes are associated with common mechanisms. The pharmacological relevance of these interactions is discussed. These data help to differentiate the structural requirements of these agents to act on GABAergic neurotransmission versus nonsteroidal anti-inflammatory effects.

Expression of heteromeric glycine receptor-channels in rat spinal cultures and inhibition by neuroactive steroids.

Ionotropic glycine receptors were studied in cultured spinal cord neurons prepared from 17-day-old rat embryos, using whole-cell patch clamp electrophysiology. Glycine receptors of 3-17 days in vitro were characterized via subtype-specific channel blockade by micromolar picrotoxin and cyanotriphenylborate, as well as nanomolar strychnine. Potentiation by nanomolar tropisetron indicated coexpression of beta with alpha subunits. The neuroactive steroids pregnenolone sulfate and dehydroepiandrosterone sulfate, as well as alphaxalone and its 3beta epimer betaxalone inhibited the chloride current with IC(50) values of 19, 46, 16 and 208 microM, respectively, with no potentiation. Reverse transcription polymerase chain reaction and immunocytochemistry demonstrated mRNAs and proteins of alpha1, alpha2, alpha3 and beta subunits in rat spinal cord cultures. In conclusion, neuroactive steroids, both positive and negative modulators of gamma-aminobutyric-acid(A) receptors, inhibited heteromeric glycine receptors at micromolar concentrations.

Interactions of granisetron with an agonist-free 5-HT3A receptor model.

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.

Novel 17beta-substituted conformationally constrained neurosteroids that modulate GABA A receptors.

The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.

Nanomolar allopregnanolone potentiates rat cerebellar GABAA receptors.

The ionophore function of gamma-aminobutyric acid A (GABA(A)) receptors was studied by whole-cell patch clamp electrophysiology in primary cultures of rat cerebellar cortex. Chloride currents elicited by 1 microM GABA were potentiated by allopregnanolone with a plateau of high affinity (EC(50) = 14 nM) and a peak of potentiation around 1 microM allopregnanolone. Furosemide (0.1 mM) eliminated the high affinity phase and increased the EC(50) to 685 nM. GABA(A) receptors of rat cerebellar synaptosomal membranes were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB). Allopregnanolone displaced [(3)H]EBOB binding with IC(50) = 320 nM. The displacing potency of allopregnanolone was strongly enhanced (IC(50) = 39 nM) in the presence of 400 nM GABA and 60 nM SR 95531. Nanomolar potentiation by allopregnanolone can be associated with cerebellar GABA(A) receptors containing alpha(6), beta(2-3) and delta subunits. This might be suitable for physiological modulation of tonic inhibitory neurotransmission via extrasynaptic GABA(A) receptors in cerebellar granule cells by neurosteroids.

Allosteric modulation of 5-HT3 serotonin receptors.

[(3)H]Granisetron binding to 5-HT(3) type serotonin receptors was examined in homogenates of rat forebrain and NG 108-15 cells. We have applied an allosteric model to 5-HT(3) receptor binding for the first time. Slope factors of displacement improved the modelling. Serotonin displaced [(3)H]granisetron binding with micromolar potency in forebrain and with nanomolar potency in NG 108-15 cells. Racemic and (+)verapamil, ifenprodil and GYKI-46903 were used as representative allosteric inhibitors of 5-HT(3) receptors. They displaced [(3)H]granisetron binding with great negative cooperativity (alpha>10) and exerted great negative cooperativity with serotonin binding (beta>10). Great negative cooperativity of these agents with serotonin and [(3)H]granisetron binding cannot be distinguished from dual competitive displacement. Trichloroethanol (data from literature) had no cooperativity with [(3)H]granisetron binding (alpha~1) and exhibit positive cooperativity with serotonin (beta<1) in displacement. The allosteric model can lead to a more quantitative method in vitro to develop allosteric agents for 5-HT(3) receptors.

High affinity, heterogeneous displacement of [3H]EBOB binding to cerebellar GABA A receptors by neurosteroids and GABA agonists.

Heterogeneous binding interactions of cerebellar GABA(A) receptors were investigated with GABA agonists and neurosteroids. GABA(A) receptors of rat cerebellum were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB), a convulsant radioligand. Saturation analysis revealed a homogenous, nanomolar population of [(3)H]EBOB binding. Both GABA and 5alpha-tetrahydrodeoxycorticosterone (5alpha-THDOC) displaced [(3)H]EBOB binding heterogeneously, with nanomolar and micromolar potencies. The nanomolar phase of displacement by GABA was selectively abolished by 100 microM furosemide. Physiological concentrations of allopregnanolone (8 nM) and 5alpha-THDOC (20 nM) increased the displacing effects of nanomolar GABA. GABA (0.3 microM ) and 5alpha-THDOC (0.3 microM ) potentiated the micromolar population of displacement by the other. Taurine inhibited [(3)H]EBOB binding also heterogeneously, with micromolar and millimolar potencies, and 0.3 microM 5alpha-THDOC potentiated this inhibition. 5beta-THDOC did not affect [(3)H]EBOB binding significantly but in 1 microM it antagonised selectively the nanomolar displacement by 5alpha-THDOC. [(3)H]EBOB binding to hippocampal GABA(A) receptors was inhibited by GABA and allopregnanolone with low (micromolar) potencies and with slope values higher than unity referring to allosteric interaction. High affinity displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be associated with constitutively open alpha(6)betadelta GABA(A) receptors, tonic GABAergic inhibitory neurotransmission and its modulation by physiological concentrations of neurosteroids.

Activation of ionotropic receptors and thermodynamics of binding.

The structure, thermodynamics and activation mechanism of Cys-loop ionotropic receptors such as glycine, nicotinic acetylcholine, 5-HT3-type serotonin and A-type gamma-aminobutyric acid receptors are discussed. Based on the interrelationship of receptor binding and ionophore function, a ternary displacement mechanism of binding including the activation of ionophores is outlined. This displacement model can explain the enigmatic thermodynamic discrimination of agonists versus antagonists of Cys-loop ionotropic receptors. Binding of both agonists and antagonists is exothermic while activation is endothermic driven by large increases in entropy. Closure of the binding cavities around agonists in concert with subunit rotations and/or removal of water-filled crevices between transmembrane (TM) regions can account for entropy increases. Recombinant glycine and gamma-aminobutyric acidA receptors and their point mutations support the predominant role of entropy in receptor activation.

Synthesis of tropeines and allosteric modulation of ionotropic glycine receptors.

Twenty esters of 3 alpha- and 3beta-hydroxy(nor)tropanes and two amides of 3 alpha-aminotropane were prepared with substituted benzoic acids. These (nor)tropeines inhibited [(3)H]strychnine binding to glycine receptors in synaptosomal membranes of rat spinal cord. A ternary allosteric model was applied to determine the dissociation constants (K(A)) of the tropeines having strong negative cooperativities with [(3)H]strychnine binding (alpha > 10). K(A) values about 10 nM are well below those of known allosteric agents. Low concentrations (0.1K(A)) of the (nor)tropeines potentiated the displacing effects of glycine. Positive cooperativity with glycine (beta < 1) decreased with the increase in concentration and binding affinity of tropeines. Displacing potencies were also measured for [(3)H]granisetron binding to 5-HT(3) type serotonin receptors of rat cerebral cortex. Selectivities to glycine receptors versus 5-HT(3) receptors varied within 4 orders of magnitude. Nortropeines might serve as a lead to high-affinity selective allosteric modulators of glycine receptors.