烟碱乙酰胆碱受体及其激动剂的研究进展

烟碱乙酰胆碱受体是离子门控通道型受体,主要分布于神经节细胞膜和骨骼肌细胞膜上。烟碱乙酰胆碱受体激动剂作用于其受体,在神经兴奋性方面发挥着重要的作用。本文对哺乳动物及昆虫烟碱乙酰胆碱受体结构及其作用的研究进行了总结,特别是对烟碱乙酰胆碱受体激动剂作用于哺乳动物及昆虫烟碱乙酰胆碱受体的选择性机理进行了概括,为开发高效、安全的杀虫剂提供了参考。     

Predicted ligand interactions for 3',5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response tothe binding of 3',5'-cyclic nucleotides. Members of the CNGCfamily vary as much as 100-fold in their ability to respondto cAMP and cGMP. Molecular models of the nucleotide bindingdomains of the bovine retina and catfish and rat olfactory CNGCswere built from the crystal structure of cAMP bound to catabolitegene activator protein (CAP) with AMMP, a program for molecularmechanics and dynamics. The nucleotide conformation can be predictedfrom the number of strong and weak interactions between thepurine ring and the binding site. The amino acids predictedto be important for determining the nucleotide affinity andspecificity are residues 61, 83 (mediated through a water molecule),119 and 127 (CAP sequence numbers) which interact with the purinering. These residues also dictate the conformation of the ligandin the binding pocket cGMP is preferentially bound in the synconformation in bovine retina, bovine olfactory and rat olfactoryCNGCs due to Thr83, while either conformation can bind in catfisholfactory CNGC. cAMP is predicted to bind either in syn or anticonformation, depending on the interaction with residue 119:the anti conformation is preferentially bound in olfactory CNGCs.     

Dynamic structural investigations on the torpedo nicotinic acetylcholine receptor by time-resolved photoaffinity labeling

An increasing number of high-resolution structures of membrane-embedded ion channels (or soluble homologues) have emerged during the last couple of years. The most pressing need now is to understand the complex mechanism underlying ion-channel function. Time-resolved photoaffinity labeling is a suitable tool for investigating the molecular function of membrane proteins, especially when high-resolution structures of related proteins are available. However until now this methodology has only been used on the Torpedo nicotinic acetylcholine receptor (nAChR). nAChRs are allosteric cation-selective receptor channels that are activated by the neurotransmitter acetylcholine (ACh) and implicated in numerous physiological and pathological processes. Time-resolved photoaffinity labeling has already enabled local motions of nAChR subdomains (i.e. agonist binding sites, ion channel, subunit interface) to be understood at the molecular level, and has helped to explain how small molecules can exert their physiological effect, an important step toward the development of drug design. Recent analytical and technical improvements should allow the application of this powerful methodology to other membrane proteins in the near future.     

Secondary structure prediction of the H5 pore of potassium channels

The ‘H5’ segment located between the putative fifthand sixth transmembrane helices is the most highly conservedregion in voltage-gated potassium channels and it is believedto constitute a major part of the ion conduction path (pore).Here we present a two-step procedure, comprising secondary structureprediction and hydrophobic moment profiling, to predict thestructure of this important region. Combined results from theapplication of the procedure to the H5 region of four voltage-gatedand five other K⁺ channel sequences lead to the prediction ofa ß-strand-turn-(3-strand structure for H5. The reasonsfor the application of these soluble protein methods to partsof membrane proteins are: (i) that pore-lining residues areaccessible to water and (ii) that a large enough database ofhighresolution membrane protein structures does not yet existThe results are compared with experimental results, in particularspectroscopic studies of two peptides based on the H5 sequenceof SHAKER potassium channel. The procedure developed here maybe applicable to wateraccessible regions of other membrane proteins.     

Molecular modelling of the nicotinic acetylcholine receptor transmembrane region in the open state

A model of the nicotinic acetylcholine receptor transmembrane region has been constructed which may represent the channel in its open-state. The positions of helices flanking the ion channel match those observed by electron microscopy and previously reported by others. Residues labelled, mutated or by other means known to have a strong influence on ion flux are each accessible from the lumen of the modelled channel. The model provides new insights into our current understanding of the ion channel structure, and suggests some novel explanations for the results of labelling and mutation studies such as those involving ion channel blockers and residue-dependent changes in ion selectivity.      

The crystal structure of {alpha}-bungarotoxin at 2.5 ? resolution: relation to solution structure and binding to acetylcholine receptor

We report collection of 2.5 ? resolution X-ray diffraction datafrom newly grown crystals of the rare ‘small unit cell’form of the long snake neurotoxin, -bungarotoxin. The previousmodel of the molecule has been rebuilt, and refined using least-squaremethods to a crystallographic residual of 0.24 at 2.5 ? resolution.-Bungarotoxin's crystal structure is compared with the crystalstructures of two other snake neurotoxins (cobratoxin and erabutoxin),and with its solution structure inferred from spectroscopicstudies. Significant differences include less ß-sheetin bungarotoxm's crystal structure than in solution, or in thecrystal structures of other neurotoxins, and an unusual orientationin the crystal of the invariant tryptophan. The functional,binding surface of bungarotoxin is described; it consists primarilyof hydrophobic and hydrogen-bonding groups and only a few chargedside chains. The structure is compared with experimental bindingparameters for neurotoxins.     

The evolutionary landscape of functional model proteins

To study the distinct influences of structure and function onevolution, we propose a minimalist model for proteins with bindingpockets, called functional model proteins, based on a shifted-HPmodel on a two-dimensional square lattice. These model proteinsare not maximally compact and contain an empty lattice sitesurrounded by at least three nearest neighbors, thus providinga binding pocket. Functional model proteins possess a uniquenative state, cooperative folding and tolerance to mutation.Due to the explicit functionality in these models (by design),we have been able to explore their fitness or evolutionary landscapes,as characterized by the size and distribution of homologousfamilies and by the complexity of the inter-relatedness of thefunctional model proteins. Mindful that these minimalist modelsare highly idealized and two-dimensional, functional model proteinsshould nevertheless provide a useful means for exploring theconstraints of maintaining structure and function on the evolutionof proteins.     

A model for vicilin solubility at mild acidic pH, based on homology modelling and electrostatics calculations

The crystallographic structures of jack bean canavalin and Frenchbean phaseolin have been used to construct a homology modelof the storage vicilin of cocoa. Reported molecular weightsfor cocoa storage protein subunits correlate with proteolysisat the site of a large hydrophilic insert in the mature protein.Burial of the hydrophobic amino acids on trimer formation isa strongly conserved feature in the vicilin family. Histidineresidues also sit at the monomer-monomer interfaces of the trimerand are likely to contribute to the decreased solubility ofcocoa vicilin at mild acidic pH, which is generally consideredto be caused solely by aggregation near to the isoelectric point.Electrostatic calculations suggest that such an arrangementof histidine residues in the absence of specific counterionbinding will not favour the particular geometry of trimer formationbelow neutral pH. Higher order aggregates that do not excludehistidine charge from the solvent may be favoured, aiding theprecipitation of cocoa vicilin at mild acidic pH. This suggestionis considered for the vicilin family. The hypothesis could contributeto an understanding of the pH and ionic strength dependenceof vicilin solubility in vitro, and possibly of the behaviourof vicilins in the seed storage environment     

Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study

Proline residues are commonly found in putative transbilayerhelices of many integral membrane proteins which act as transporters,channels and receptors. Intramembranous prolines are often conservedbetween homologous proteins. It has been suggested that suchintrahelical prolines provide liganding sites for cations viaexposure of the backbone carbonyl oxygen atoms of residues i-3and i-4 (relative to the proline). Molecular modelling studieshave been carried out to evaluate this proposal. Bundles ofparallel proline-kinked helices are considered as simplifiedmodels of ion channels. The energetics of K⁺ ion-helix bundleinteractions are explored. It is shown that carbonyl oxygensexposed by the proline-induced kink and at the C-terminus ofthe helices may provide cation-liganding sites. ‘Hybrid’bundles of antiparallel helices, only some of which containproline residues, are considered as models of transport proteins.Again, praline-exposed carbonyi oxygens are shown to be capableof liganding cations. The roles of -helix dipoles and of thegeometry of helix packing are considered in relation to cation-bundleinteractions. Implications with respect to modelling of ionchannel and transport proteins are discussed     

Molecular dynamics studies of AChBP with nicotine and carbamylcholine: the role of water in the binding pocket

The acetylcholine-binding protein (AChBP) is homologous to the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) and other members of the Cys-loop family of neurotransmitter receptors. The high-resolution X-ray structures of AChBP mean it has been used as a model from which to understand agonist and antagonist binding to nAChRs. We present here a molecular dynamics (MD) study of AChBP with nicotine and carbamylcholine bound. Our results suggest that the ligand imposes rigidity on the binding pocket residues. The simulations also suggest that the protein undergoes breathing motions with respect to the five-fold axis, a motion that has been postulated to be related to gating in the nAChR. We analyzed the behaviour of the water molecules in and around the binding site and found that they occupied five distinct sites within the binding pocket. Water occupied these sites in the absence of ligand, but the presence of ligand increased the probability that a water molecule would be found in these sites. Finally, we demonstrate how the positions of these waters might be used in the design of new ligands by comparing the positions of these sites with other recent structures.     

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain

Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine-cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.     

A 3D model of the {delta} opioid receptor and ligand-receptor complexes

A model for the 3D structure of the transmembrane domain ofthe opioid receptor was predicted from the sequence divergenceanalysis of 42 sequences of G-protein coupled peptide hormonereceptors belonging to the opioid, somatostatin and angiotensinreceptor families. No template was used in the prediction steps,which include multiple sequence alignment, calculation of avariability profile of the aligned sequences, use of the variabilityprofile to identify the boundaries of transmembrane regions,prediction of their secondary structure, optimization of thepacking shape in a helix bundle, prediction of side chain conformationsand structural refinement The general shape of the model issimilar to that of the low resolution rhodopsin structure inthat the TM3 and TM7 helices are most buried in the bundle andthe TM1 and TM4 helices are most exposed to the lipid phase.An initial assessment of this model was made by determiningto what extent a binding site identified using four structurallydisparate high affinity opioid ligands was consistent withknown mutational studies. With the assumption that the pro-tonatedamine nitrogen, a feature common to all opioid ligands, interactswith the highly conserved Aspl27 in TM3, a pocket was foundthat satisfied the criteria of complementarity to the requirementsfor receptor recognition for these four diverse ligands, two selective antagonists (the fused ring naltrindole and the peptideTyr-Tic-Phe-Phe-NH₂) and the two agonists lofentanil and BW373U86deduced from previous studies of the ligands alone. These ligandscould be accommodated in a similar region of the receptor. Thereceptor binding site identified in the optimized complexescontained many residues in positions known to affect ligandbinding in G-protein coupled receptors. These results also allowedidentification of key residues as candidates for point mutationsfor further assessment and refinement of this model as wellas preliminary indications of the requirements for recognitionof this receptor.     

Use of a quantitative structure-property relationship to design larger model proteins that fold rapidly

A quantitative structure–property relationship (QSPR)was used to design model protein sequences that fold repeatedlyand relatively rapidly to stable target structures. The specificmodel was a 125-residue heteropolymer chain subject to MonteCarlo dynamics on a simple cubic lattice. The QSPR was derivedfrom an analysis of a database of 200 sequences by a statisticalmethod that uses a genetic algorithm to select the sequenceattributes that are most important for folding and a neuralnetwork to determine the corresponding functional dependenceof folding ability on the chosen attributes. The QSPR dependson the number of anti-parallel sheet contacts, the energy gapbetween the native state and quasi-continuous part of the spectrumand the total energy of the contacts between surface residues.Two Monte Carlo procedures were used in series to optimize boththe target structures and the sequences. We generated 20 fullyoptimized sequences and 60 partially optimized control sequencesand tested each for its ability to fold in dynamic MC simulations.Although sequences in which either the number of anti-parallelsheet contacts or the energy of the surface residues is non-optimalare capable of folding almost as well as fully optimized ones,sequences in which only the energy gap is optimized fold markedlymore slowly. Implications of the results for the design of proteinsare discussed.     

A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization

A mechanism by which ligand binding to the extracellular domainof a growth factor receptor causes activation of its cytoplasmictyrosine kinase domain is that binding promotes receptor dimerization.Recently we proposed a model in which dimerization of the transmembrane-helices in one member of this family, rat neu, is mediatedby the presence of three specific residues. This paper showsthat a similar sequence motif is observed in 18 of the 20 transmembrane-helices of the tyrosine kinase family of growth factor receptors.The motif encompasses a five residue segment in which position0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro),P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 onlythe smallest side chains (Gly or Ala). In addition other featuresof the transmembrane sequences are reported. It is concludedthat the dimerization of transmembrane -helices may be a generalmechanism of tyrosine kinase activation in this family of growthfactor receptors.     

Functional implications of the modeled structure of maspin

The tumor suppressor maspin (mammary-specific serpin) is anunstable serpin that does not undergo the stressed to relaxedtransition typical of proteinase inhibitory serpins and, consequently,is not likely to function as a serine proteinase inhibitor.This suggests that the positioning and configuration of thereactive site loop (RSL) of maspin are likely to resemble thoseof ovalbumin, the best studied non-inhibitory serpin. Accordingly,the tertiary structure of maspin has been modeled on the crystalstructure of native ovalbumin. Biochemical data and the modeledtheoretical structure of maspin reveal the absence of disulfidebonds in the molecule and the presence of an unstable RSL thatadopts a distorted helical structure. We confirm that the RSLis extremely sensitive to limited proteolysis and suggest thatthis may provide a structural basis for the proteolytic inactivationof maspin, a process that is likely to modulate the activityof maspin in biological systems.     

Influenza virus M2 protein: a molecular modelling study of the ion channel

The influenza A M₂ protein forms cation-selective ion channelswhich are blocked by the anti-influenza drug amantadine. A molecularmodel of the M₂ channel is presented in which a bundle of fourparallel M₂ transbilayer helices surrounds a central ion-permeablepore. Analysis of helix amphipathicity was used to aid determinationof the orientation of the helices about their long axes. Thehelices are tilted such that the N-terminal mouth of the poreis wider than the C-terminal mouth. The channel is lined byresidues V27, S31 and I42. Residues D24 and D44 are locatedat opposite mouths of the pore, which is narrowest in the vicinityof I42. Energy profiles for interaction of the channel withNa⁺, amantadine-H⁺ and cyclopentylamine-H⁺ are evaluated. Theinteraction profile for Na⁺ exhibits three minima, one at eachmouth of the pore, and one in the region of residue S31. Theamantadine-H⁺ profile exhibits a minimum close to S31 and abarrier near residue I42. This provides a molecular model foramantadine-H⁺ block of M₂ channels. The profile for cyclopentylamine-H⁺does not exhibit such a barrier. It is predicted that cyclopentyl-amine-H⁺will not act as an M₂ channel blocker.     

Predicting the point at which transmembrane helices protrude from the bilayer: a model of the antenna complexes from photosynthetic bacteria

We describe a method for predicting the point at which a transmembranehelix leaves the bilayer and enters the more polar region ofthe aqueous exterior. This is achieved by comparing the relativedirections of the hydrophobic and internal faces of the transmembranehelices which should be opposite for the regions within thebilayer but equivalent for the regions on the outside. Thisinformation provides a strong constraint in the process of modellingmembrane proteins. We go on to use the approach to model themonomers of the bacterial light-harvesting antenna complexes.This information is then combined with some preliminary crystallographkdata and biochemical results to produce a 3-D model of a tetramer.     

A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator

The homozygous deletion of the phenylalanine at position 508 (DeltaPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and DeltaPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the 'folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the DeltaPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against CF.     

Coiled-coil molecular recognition: directed solution assembly of receptor ectodomains

The high affinity interleukin-2 (IL-2) receptor is composedof at least three cell surface proteins (, ß and subunits), each of which is independently capable of ligandbinding. Physiologically, these subunits cooperate to form dimericand trimeric complexes that efficiently capture IL-2 and transmitthe signal across the membrane. The knowledge of how each subunitfunctions with respect to ligand capture, signal transmissionand internalization is essential for the development of ligand-basedIL-2 agonists and antagonists, as well as receptor-related therapeuticand diagnostic reagents. Only one of the subunits (p55 or chain)is capable of interacting with ligand in solution in a mannerthat resembles cell surface binding. To generate soluble multhnerkcomplexes of the IL-2 receptor subunits that may bind ligandin solution in a fashion that mimics the same receptor complexeson the cell surface, we have added recognition sequences (coiled-coilheptad repeats) to the ectodomains of the individual receptors.Here we describe the expression and characterization of a prototypeIL-2ß receptor ectodomain-coiled-coil fusion proteinand demonstrate that this is a feasible approach to the preparationof cytokine receptor solution complexes.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis

The Escherichia coli aspartate receptor is a dimer with twotransmembrane sequences per monomer that connect a periplasmicligand binding domain to a cytoplasmic signaling domain. Themethod of 'hydrophobic-biased' random mutagenesis, that we describehere, was used to construct mutant aspartate receptors in whicheither the entire transmembrane sequence or seven residues nearthe center of the transmembrane sequence were replaced withhydrophobic and polar random residues. Some of these receptorsresponded to aspartate in an in vivo chemotaxis assay, whileothers did not. The acceptable substitutions included hydrophobicto polar residues, small to larger residues, and large to smallerresidues. However, one mutant receptor that had only a few hydrophobicsubstitutions did not respond to aspartate. These results addto our understanding of sequence specificity in the transmembraneregions of proteins with more than one transmembrane sequence.This work also demonstrates a method of constructing familiesof mutant proteins containing random residues with chosen characteristics.