ABSTRACT
X-ray crystallography, structural bioinformatics and computational chemistry have become important techniques in the discovery and development of effective and safe new drugs. From a drug discovery point of view, membrane proteins are among the most interesting molecular targets, but the current knowledge about detailed 3D structures of membrane proteins is sparse. Homology modeling techniques may provide structural knowledge about membrane proteins and their interactions with drugs and other molecules. The neurotransmitter sodium symporters (NSS) are the molecular targets of many pharmacologically active substances, and we have used three different secondary transporters as templates for modeling the NSS proteins DAT, NET and SERT. The first template was based on the electron density projection map of the Escherichia coli Na+/H+ antiporter (NhaA), while later the X-ray structure of Lac Permease (symporter) was used as a template. The helical architectures of these templates have a lot in common, and models based on both could contribute with structural explanations of several experimental studies in spite of low homology with NSS proteins. In 2005 the crystal structure of a bacterial homologue of the human monoamine neurotransmitter transporter Aquifex aeolicus (LeuTAa) was reported. This structure was the first experimental structure of a NSS family member, and represented a breakthrough for homology modeling of pharmacological important NSS proteins. Since then several X-ray structures LeuTAa in complex with pharmacologically important compounds have been published. Homology models of NSS proteins, combined with site-directed mutagenesis data, have identified ligand binding sites and contributed with important knowledge for new drug development.
Subject(s)
Membrane Transport Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli Proteins/chemistry , Humans , Membrane Transport Proteins/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Neurotransmitter Transport Proteins/chemistry , Neurotransmitter Transport Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Protein Conformation , Serotonin Plasma Membrane Transport Proteins/chemistry , Sodium-Hydrogen Exchangers/chemistryABSTRACT
A three-dimensional electron density projection map of the ion-coupled membrane protein Escherichia coli Na+/H+ antiporter (NhaA) was recently published. Based on this projection map, and previous biophysical studies determining the assignment of the 12 transmembrane alpha-helices (TMHs), a three-dimensional molecular model of the NhaA was constructed, using interactive molecular graphics and energy calculations. The diuretic drug, amiloride, was docked into the model and putative interacting amino acids were identified. The model suggests that the pH dependent activity of NhaA may be explained by charge changes in the intracellular loop between TMH8 and TMH9 which alter the positions of TMHs 4, 5 and 11 relative to each other, such that a pore area of the transporter protein is opened.
Subject(s)
Bacterial Proteins/chemistry , Computer Simulation , Escherichia coli Proteins , Models, Molecular , Sodium-Hydrogen Exchangers/chemistry , Escherichia coli , Protein Structure, TertiaryABSTRACT
The human serotonin transporter is the molecular target for selective serotonin reuptake inhibitor drugs which are being used for treatment of depression. A three-dimensional model of the membrane spanning parts of the transporter was constructed. The transporter was assumed to consist of 12 transmembrane alpha-helices. The model was based on published experimental data of cocaine binding to mutant transporters, amino acid sequence analysis, and interactive molecular graphics. The model suggests that a high affinity cocaine binding site is situated in a region of the model where Asp98 acts like an anchor, while a putative low affinity site is situated in another region with Glu508 as the anchoring amino acid. A series of docking experiments with various reuptake inhibitors were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. Experiments involving molecular mapping of ligand binding areas may benefit from using the current model in experimental design. From the current model, several amino acids were proposed as prime candidates for mutagenesis and subsequent ligand binding studies. Also for evaluation of results from site directed mutagenesis experiments with SERT and similar transporters we assume the model will be helpful.
Subject(s)
Carrier Proteins/chemistry , Membrane Glycoproteins/chemistry , Membrane Transport Proteins , Nerve Tissue Proteins , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cocaine/metabolism , Computer Simulation , Drug Design , Humans , In Vitro Techniques , Ligands , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Structure, Secondary , Serotonin Plasma Membrane Transport Proteins , Selective Serotonin Reuptake Inhibitors/chemistry , Selective Serotonin Reuptake Inhibitors/pharmacology , ThermodynamicsABSTRACT
A World-Wide Web service has been constructed to assist the development of force field parameters extending the AMBER force fields. This service extracts parameters from the standard AMBER force field parameter databases. From a Web-based interface the user can choose between bond, angle and torsional parameters with certain constraints on element and/or atom hybridization. The software constructed for the purpose of finding appropriate parameters will locate standard AMBER force field parameters matching the user specification. For bond and angle parameters a scatter plot of the reference values against force constants is provided. This service has been produced to assist in extraction and evaluation of parameters that may be useful for molecules other than proteins and nucleic acids.
