ABSTRACT
Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (P(i)) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters.
Subject(s)
Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Sodium Chloride/chemistry , Amino Acids/genetics , Amino Acids/metabolism , Binding Sites , Computer Simulation , Crystallography, X-Ray , Kinetics , Membrane Transport Proteins/genetics , Models, Molecular , Mutation/genetics , Protein Structure, Tertiary , ProtonsABSTRACT
Tricyclic antidepressants exert their pharmacological effect-inhibiting the reuptake of serotonin, norepinephrine, and dopamine-by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.
Subject(s)
Antidepressive Agents, Tricyclic/metabolism , Bacterial Proteins/metabolism , Desipramine/metabolism , Neurotransmitter Uptake Inhibitors/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Amino Acid Sequence , Animals , Antidepressive Agents, Tricyclic/chemistry , Bacterial Proteins/chemistry , Binding Sites , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Cell Line , Conserved Sequence , Crystallography, X-Ray , Desipramine/chemistry , Dopamine/chemistry , Dopamine/metabolism , Dopamine Uptake Inhibitors/chemistry , Dopamine Uptake Inhibitors/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Humans , Leucine/chemistry , Leucine/metabolism , Models, Molecular , Molecular Sequence Data , Neurotransmitter Uptake Inhibitors/chemistry , Norepinephrine/chemistry , Norepinephrine/metabolism , Norepinephrine Plasma Membrane Transport Proteins/antagonists & inhibitors , Norepinephrine Plasma Membrane Transport Proteins/chemistry , Norepinephrine Plasma Membrane Transport Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Serotonin/chemistry , Serotonin/metabolism , Selective Serotonin Reuptake Inhibitors/chemistry , Selective Serotonin Reuptake Inhibitors/metabolismABSTRACT
Although codon usage is often represented by a 61-dimensional vector, the ability of determining the codon bias in a gene relies on a uni-dimensional vector which measures the total bias in usage of synonymous codons. Codon usage is receiving more and more focus because codon biases might be valuable tools to predict and optimize gene/protein expression. How good any of these measures is for correlating codon usage with gene and protein expression has yet to be investigated. In this study, we correlated gene transcript levels in Escherichia coli with codon usage, using a number of different codon bias measures. We found that there is a significant correlation between transcript levels and codon bias measures, suggesting that these measures can be used to assess or predict gene expression. The codon bias measure performing best in this context was the codon adaptation index.
