RESUMEN
Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/química , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Genoma Bacteriano , Cinética , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
Until now, voltage-gated Ca(2+) channel proteins have been found only in eukaryotes. Here we report that a gene recently discovered in the eubacterium Bacillus halodurans codes for a protein closely related to eukaryotic Ca(2+) channels, but that has only one 6-transmembrane-segement (6TM) motif, instead of four, in its pore-forming subunit. This is supported by the comparison of consensus sequences, which, along with the patterns of residue conservation, indicates a similar structure in the membrane to voltage-gated K(+) channels. From this we hypothesize that Ca(2+) channels originally evolved in bacteria, and that the specific eubacteria protein highlighted here is an ideal candidate for structure determination efforts.
Asunto(s)
Canales de Calcio/química , Activación del Canal Iónico , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Fluctuations about the native conformation of proteins have proven to be suitably reproduced with a simple elastic network model, which has shown excellent agreement with a number of different properties for a wide variety of proteins. This scalar model simply investigates the magnitudes of motion of individual residues in the structure. To use the elastic model approach further for developing the details of protein mechanisms, it becomes essential to expand this model to include the added details of the directions of individual residue fluctuations. In this paper a new tool is presented for this purpose and applied to the retinol-binding protein, which indicates enhanced flexibility in the region of entry to the ligand binding site and for the portion of the protein binding to its carrier protein.
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Proteínas/química , Anisotropía , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Cristalografía por Rayos X , Elasticidad , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas de Unión al Retinol/química , TermodinámicaRESUMEN
BACKGROUND: Prior to this report, members of the inward rectifier family, or Kir, have been found only in eukaryotes. Like most K+ channels, the pore-forming part of the protein is formed by four identical, or closely related, subunits. Each subunit contains a transmembrane M1-P-M2 motif that is followed by a relatively large C-terminus region unique to Kir's. RESULTS: In searching unfinished microbial genomes for K+ channels, we identified five sequences in the prokaryote Burkholderia pseudomallei, Burkholderia cepacia, Burkholderia fungorum LB400, Magentospirillum magnetotacticum, and Nostoc Punctiforme genomes that code for proteins whose closest relatives in current sequence databases are eukaryote Kir's. The sequence similarity includes the C-terminus portion of Kir's, for which there are no other close homologs in current prokaryote sequences. Sequences of the pore-forming P and M2 segments of these proteins, which we call KirBac, is intermediate between those of eukaryotic Kir's and several other K+ channel families. CONCLUSIONS: Although KirBac's are more closely related to Kir's than to other families of K+ channels, the intermediate nature of their pore-forming P and M2 segments suggests that they resemble an ancestral precursor to the eukaryotic Kir's. The similarity of KirBac to the bacterial KcsA channel, whose transmembrane structure has been solved, helps align Kir's with KcsA. KirBac's may assist in solving the three-dimensional structure of a member of the Kir family since bacterial membrane proteins are more easily expressed in the quantities necessary for crystallography.
Asunto(s)
Burkholderia/genética , Canales de Potasio de Rectificación Interna/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Burkholderia cepacia/genética , Burkholderia pseudomallei/genética , Cianobacterias/genética , Bases de Datos Genéticas , Genoma Bacteriano , Datos de Secuencia Molecular , Familia de Multigenes , Canales de Potasio de Rectificación Interna/química , Estructura Terciaria de Proteína/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Evidence is presented that the transmembrane KdpA subunit of the high affinity K(+)-translocating P-type Kdp-ATPase is evolutionarily derived from the superfamily of 2TM-type K(+) channels in bacteria. This extends a previous study relating the K(+) channels to the KtrAB, Trk, Trk1,2, and HKT1 K(+) symporter superfamily of both prokaryotes and eukaryotes. Although the channels are formed by four single-MPM motif subunits, the transmembrane KdpA subunit and the transmembrane subunit of the symporter proteins are postulated to have four corresponding MPM motifs within a single sequence. Analysis of 17 KdpA sequences reveals a pattern of residue conservation similar to that of the symporters and channels, and consistent with the crystal structure of the KcsA K(+) channel. In addition, the most highly conserved residues between the families, specifically the central glycines of the P2 segments, are those previously identified as crucial for the property of K(+)-selectivity that is common to each protein. This hypothesis is consistent with an experimental study of mutations that alter K(+) binding affinity of the Kdp transporter. Although most of the results of a previous study of the transmembrane topology of KdpA are consistent with the 4-MPM model, the one deviation can be explained by a plausible change in the structure due to the experimental method.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Portadoras/química , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Canales de Potasio/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Portadoras/metabolismo , Membrana Celular/ultraestructura , Secuencia Conservada , Bases de Datos como Asunto , Evolución Molecular , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Potasio/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The hypothesis is presented that at least four families of putative K(+) symporter proteins, Trk and KtrAB from prokaryotes, Trk1,2 from fungi, and HKT1 from wheat, evolved from bacterial K(+) channel proteins. Details of this hypothesis are organized around the recently determined crystal structure of a bacterial K(+) channel: i. e., KcsA from Streptomyces lividans. Each of the four identical subunits of this channel has two fully transmembrane helices (designated M1 and M2), plus an intervening hairpin segment that determines the ion selectivity (designated P). The symporter sequences appear to contain four sequential M1-P-M2 motifs (MPM), which are likely to have arisen from gene duplication and fusion of the single MPM motif of a bacterial K(+) channel subunit. The homology of MPM motifs is supported by a statistical comparison of the numerical profiles derived from multiple sequence alignments formed for each protein family. Furthermore, these quantitative results indicate that the KtrAB family of symporters has remained closest to the single-MPM ancestor protein. Strong sequence evidence is also found for homology between the cytoplasmic C-terminus of numerous bacterial K(+) channels and the cytoplasm-resident TrkA and KtrA subunits of the Trk and KtrAB symporters, which in turn are homologous to known dinucleotide-binding domains of other proteins. The case for homology between bacterial K(+) channels and the four families of K(+) symporters is further supported by the accompanying manuscript, in which the patterns of residue conservation are demonstrated to be similar to each other and consistent with the known 3D structure of the KcsA K(+) channel.
Asunto(s)
Proteínas de Transporte de Catión , Evolución Molecular , Canales de Potasio/genética , Proteínas de Saccharomyces cerevisiae , Simportadores , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Fenómenos Biofísicos , Biofisica , Proteínas Portadoras/genética , Secuencia Conservada , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Proteínas de Plantas/genética , Canales de Potasio/química , Canales de Potasio/metabolismo , Conformación Proteica , Homología de Secuencia de Aminoácido , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Three-dimensional computer modeling is used to further investigate the hypothesis forwarded in the accompanying paper of an evolutionary relationship between four related families of K(+) sympoter proteins and the superfamily of K(+) channel proteins. Atomic-scale models are developed for the transmembrane regions of one member from each of the three more distinct symporter families, i.e., a TrkH protein from Escherichia coli, a KtrB protein from Aquifex aeolicus, and a Trk1,2 protein from Schizosaccharomyces pombe. The portions of the four consecutive M1-P-M2 motifs in the symporters that can be aligned with K(+) channel sequences are modeled directly from the recently determined crystal structure of the KcsA K(+) channel from Streptomyces lividans. The remaining portions are developed using our previously accumulated theoretical modeling criteria and principles. Concurrently, the use of these criteria and principles is further supported by the now verified predictions of our previous K(+) channel modeling efforts and the degree to which they are satisfied by the known structure of the KcsA protein. Thus the observed ability of the portions of the symporter models derived from the KcsA crystal structure to also satisfy the theoretical modeling criteria provides additional support for an evolutionary link with K(+) channel proteins. Efforts to further satisfy the criteria and principles suggest that the symporter proteins from fungi and plants (i.e., Trk1,2 and HKT1) form dimeric and/or tetrameric complexes in the membrane. Furthermore, analysis of the atomic-scale models in relation to the sequence conservation within and between the protein families suggests structural details for previously proposed mechanisms for the linked symport of K(+) with Na(+) and H(+). Suggestions are also given for experiments to test these structures and hypotheses.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/química , Proteínas de Transporte de Catión , Modelos Moleculares , Canales de Potasio/química , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Fenómenos Biofísicos , Biofisica , Proteínas Portadoras/genética , Electroquímica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Canales de Potasio/genética , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.
Asunto(s)
Quimiocinas/metabolismo , Mapeo Epitopo , Receptores CCR5/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Línea Celular , Simulación por Computador , Citometría de Flujo , Productos del Gen env/metabolismo , VIH-1 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores CCR5/inmunología , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32-36 degrees, whereas wt p53 bends it by 51-57 degrees. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by approximately 35 degrees and approximately 70 degrees, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.
Asunto(s)
ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
A large collaborative, multidisciplinary effort involving many research laboratories continues which uses indirect methods of molecular biology and membrane biophysics to analyze the three-dimensional structures and functional mechanisms of K+ channels. This work also extends to the distant relatives of these channels, including the voltage-gated Na+ and Ca2+ channels. The role that our group plays in this process is to combine the information gained from experimental studies with molecular modeling techniques to generate atomic-scale structural models of these proteins. The modeling process involves three stages which are summarized as: (I) prediction of the channel sequence transmembrane topology, including the functionality and secondary structure of the segments; (II) prediction of the relative positions of the transmembrane segments, and (III) filling in all atoms of the amino acid residues, with conformations for energetically stabilized interactions. Both physiochemical and evolutionary principles (including sequence homology analysis) are used to guide the development. In addition to testing the steric and energetic feasibilities of different structural hypotheses, the models provide guidance for the design of new experiments. Structural modeling also serves to "fill in the gaps" of experimental data, such as predicting additional residue interactions and conformational changes responsible for functional processes. The modeling process is currently at the stage that experimental studies have definitely confirmed most of our earlier predictions about the transmembrane topology and functionality of different segments. Additionally, this report describes the detailed, three-dimensional models we have developed for the entire transmembrane region and important functional sites of the voltage-gated Shaker K+ channel in the open, closed, and inactivated conformations (including the ion-selective pore and voltage-sensor regions). As part of this effort, we also describe how our development of structural models for many of the other major K+ channel families aids in determining common structural motifs. As an example, we also present a detailed model of the smaller, bacterial K+ channel from Streptomyces lividans. Finally, we discuss strategies for using newly developed experimental methods for determining the structures and analyzing the functions of these channel proteins.
Asunto(s)
Modelos Moleculares , Canales de Potasio/química , Conformación Proteica , Secuencia de Aminoácidos , Animales , Simulación por Computador , Células Eucariotas/química , Activación del Canal Iónico , Datos de Secuencia Molecular , Canales de Potasio/fisiología , Células Procariotas/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia ShakerRESUMEN
This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.
Asunto(s)
Fusión de Membrana/fisiología , Proteínas Virales de Fusión/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Consenso , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Orthomyxoviridae/genética , Orthomyxoviridae/fisiología , Unión Proteica , Alineación de Secuencia , Homología de Secuencia , Relación Estructura-Actividad , Proteínas Virales de Fusión/química , Fenómenos Fisiológicos de los VirusRESUMEN
Molecular dynamics simulations are used to model the transfer thermodynamics of krypton from the gas phase into water. Extra long, nanosecond simulations are required to reduce the statistical uncertainty of the calculated "solvation" enthalpy to an acceptable level. Thermodynamic integration is used to calculate the "solvation" free energy, which together with the enthalpy is used to calculate the "solvation" entropy. A comparison series of simulations are conducted using a single Lennard-Jones sphere model of water to identify the contribution of hydrogen bonding to the thermodynamic quantities. In contrast to the classical "iceberg" model of hydrophobic hydration, the favorable enthalpy change for the transfer process at room temperature is found to be due primarily to the strong van der Waals interaction between the solute and solvent. Although some stabilization of hydrogen bonding does occur in the solvation shell, this is overshadowed by a destabilization due to packing constraints. Similarly, whereas some of the unfavorable change in entropy is attributed to the reduced rotational motion of the solvation shell waters, the major component is due to a decrease in the number of positional arrangements associated with the translational motions.
Asunto(s)
Criptón/química , Modelos Químicos , Calorimetría , Gases , Cinética , Modelos Estadísticos , Soluciones , Termodinámica , Factores de Tiempo , AguaRESUMEN
A new generation of structural models were developed of the outer vestibule and ion-selective portion of the voltage-gated Shaker K+ channel. Some features of these models are similar to those that we have developed previously [Durrel S. R. and Guy H. R. (1992) Biophys. J. 62, 238-250; Guy H. R. (1990) In Monovalent Cations in Biological Systems (Pasternak C. A., Ed.), pp. 31-58, CRC Press, Boca Raton, FL; Guy H. R. and Durell S. R. (1994) In Molecular Evolution of Physiological processes (Fambrough D., Ed.), pp. 197-212, The Rockefeller University Press, NY; Guy H. R. and Durell S. R. (1995) In Ion Channels and Genetic Diseases (Dawson D., Ed.), pp. 1-16, The Rockefeller University Press, NY] and other features were modified to make the models more consistent with recent experimental findings. The first part of the P segment is postulated, as always, to form a short alpha helix that spans only the outer portion of the membrane. The helix is tilted so that its C-terminal is nearer the pore than its N-terminal. The latter part of the P segment, P2, is postulated to have a relatively elongated conformation that is positioned approximately parallel to the axis of the pore. Four of the P2 segments assemble to form an ion-selective region that has two narrow regions; one formed by the Y445 side-chains at the outer entrance of the pore and one formed by the backbone of the T442 residues near the innermost part of the P segments. The S6 segment is postulated to form two alpha helices. The first S6 helix packs next to the P segments in our models. The NMR structures of two scorpion toxins, charybdotoxin and agitoxin 2, have been docked into the models of the outer vestibules. The shape of the outer vestibule has been modeled so that specific toxin-channel residue-residue interactions correspond to those that have been identified experimentally.
Asunto(s)
Modelos Moleculares , Canales de Potasio/química , Estructura Secundaria de Proteína , 4-Aminopiridina/metabolismo , Caribdotoxina , Activación del Canal Iónico , Mutación , Canales de Potasio/genética , Canales de Potasio/metabolismo , Venenos de Escorpión/metabolismo , Canales de Potasio de la Superfamilia Shaker , Tetraetilamonio , Compuestos de Tetraetilamonio/metabolismo , Toxinas Biológicas/metabolismoRESUMEN
The structure determination of voltage-gated channels by the combination of molecular modeling and mutagenesis experiments is a long term, iterative process. As such, the models should be considered as a work in progress, with changes expected as more data becomes available. The primary role of the models is that they assimilate the known data and provide ideas for further experiments to elucidate the real structures. Although the models presented here have already gone through two or three cycles of development and testing, many aspects remain tentative. Perhaps the most significant result so far is that the P segment was experimentally confirmed to form the ion-selective part of the channel. In a subsequent cycle of testing and modeling, the specific residues responsibility for Na+ and Ca2+ selectivity have been identified and the selectivity filter of K+ channels is now predicted to be formed by the side chains, but rather by the carbonyl oxygens of the conserved Gly-Tyr-Gly sequence backbone. As another example, the 9p residue of the P segment of K+ channels was originally modeled as either being buried in the protein or accessible channels was originally modeled as either being buried in the protein or accessible from inside the cell only. However, once mutation of this residue to histidine was found to affect blockade by extracellular TEA, protons, Zn2+ and histidine reagents (DeBiasi et al., 1993), the models were updated to have this and the hydrophilic residues in the first part of P form a helix that comprises part of the extracellular, outer vestibular of the pore. While this motif was used also for Na+ and Ca2+ pore models (see Fig. 2) where the putative helices are amphipathic, it remains to be verified. Modeling of the size and shape of the outer vestibule of K+ channels was also aided by the data for the binding of CTX in the extracellular entrance to the pore. Similarly, experiments with peptide toxins such as mu and omega conotoxins may prove useful in modeling the outer vestibules of the Na+ and/or Ca2+ channels. While important advances have been made, it is important to realize that these approaches are still very new. In the future we are likely to see improvements on both the theoretical and experimental sides which will greatly advance the process.(ABSTRACT TRUNCATED AT 400 WORDS)
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Canales de Calcio/fisiología , Modelos Moleculares , Canales de Potasio/fisiología , Canales de Sodio/fisiología , Animales , Sitios de Unión , Transporte IónicoRESUMEN
Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimer's disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimer's disease, then the models may be helpful in designing counteracting drugs.
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Péptidos beta-Amiloides/química , Canales Iónicos/química , Modelos Moleculares , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/genética , Fenómenos Biofísicos , Biofisica , Humanos , Canales Iónicos/genética , Membrana Dobles de Lípidos/química , Datos de Secuencia Molecular , Estructura Molecular , Ovillos Neurofibrilares/química , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
Molecular modeling and mutagenesis analysis of voltage-gated channels have succeeded in identifying much of the topology of the proteins and in identifying which sequential segments are involved in functional mechanisms such as activation gating, inactivation gating, ion selectivity, and ligand binding. Efforts are currently underway to use these methods to model the protein structure and functional mechanisms more precisely. The experimental and theoretical efforts are dependent to a considerable extent upon information obtained by comparing homologous sequences. Although the fine details of models developed in this manner are unlikely to be as correct as models developed from x-ray crystallography and NMR, they still may contribute substantially to our understanding of the structure and function of these important proteins.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/genética , Canales Iónicos/fisiología , Secuencia de Aminoácidos , Animales , Electrofisiología , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Atomic-scale computer models were developed for how cecropin peptides may assemble in membranes to form two types of ion channels. The models are based on experimental data and physiochemical principles. Initially, cecropin peptides, in a helix-bend-helix motif, were arranged as antiparallel dimers to position conserved residues of adjacent monomers in contact. The dimers were postulated to bind to the membrane with the NH2-terminal helices sunken into the head-group layer and the COOH-terminal helices spanning the hydrophobic core. This causes a thinning of the top lipid layer of the membrane. A collection of the membrane bound dimers were then used to form the type I channel structure, with the pore formed by the transmembrane COOH-terminal helices. Type I channels were then assembled into a hexagonal lattice to explain the large number of peptides that bind to the bacterium. A concerted conformational change of a type I channel leads to the larger type II channel, in which the pore is formed by the NH2-terminal helices. By having the dimers move together, the NH2-terminal helices are inserted into the hydrophobic core without having to desolvate the charged residues. It is also shown how this could bring lipid head-groups into the pore lining.
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Péptidos Catiónicos Antimicrobianos , Hormonas de Insectos/química , Proteínas de Insectos , Canales Iónicos/química , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Biofisica , Simulación por Computador , Datos de Secuencia Molecular , Conformación Proteica , TermodinámicaRESUMEN
We have examined the ion channel forming properties of magainin 2 by incorporating the peptide into artificial lipid bilayers held under voltage clamp. Magainin 2 increased lipid bilayer conductance in a concentration dependent manner with a Hill coefficient of 1.7. The magainin 2 conductance was selective for monovalent cations over anions with a ratio of 5:1 and had both voltage-sensitive and -insensitive components. Two structurally related but antibiotically less potent analogues, magainin 1 and Z-12, also increased lipid bilayer conductance with a similar ion selectivity but these peptides were less potent than magainin 2. We propose that the weak cation selectivity of the magainin channels can be accounted for by the inclusion of negatively charged lipids in the channel complex and suggest two possible structures for such a channel. The ionophoric properties of these peptides are likely to be proximal to their antibiotic activities.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos , Canales Iónicos/fisiología , Membrana Dobles de Lípidos/metabolismo , Péptidos/farmacología , Proteínas de Xenopus , Animales , Antiinfecciosos/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Canales Iónicos/metabolismo , Magaininas , Modelos Biológicos , Péptidos/metabolismoRESUMEN
Recent mutagenesis experiments have confirmed our hypothesis that a segment between S5 and S6 forms the ion selective portion of voltage-gated ion channels. Based on these and other new data, we have revised previous models of the general folding pattern of voltage-gated channel proteins and have developed atomic scale models of the entire transmembrane region of the Shaker A K+ channel. In these models, the ion selective region is a beta-barrel that spans the outer half of the membrane. The inner half of the pore is larger. The voltage-dependent conformational changes of activation gating are modeled to occur by the "helical screw" mechanism, in which the four S4 segments move along and rotate about their axes. These changes are followed by a voltage-independent conformational change, in which the segments linking S4 to S5 move from blocking the intracellular entrance of the pore to forming part of the lining of the large inner portion of the pore. The NH2-terminal of the protein was modeled as an alpha-helix that plugs the intracellular half of the pore to inactivate the channel.