Dopamine receptors Dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions abnormalities in its regulation have been implicated in neuropsychiatric and substance use disorders. That dopamine D3 receptor (D3R) expression is elevated in response to drugs of abuse, has prompted efforts toward the development of D3R-selective agents for the treatment of drug addiction. Inhibition of D3R may be less prone to causing motor side effects that can result from D2R blockade. In addition to inhibiting the behavioral effects of cocaine, D3R partial agonists may lead to better compliance in treating addiction. Thus in contrast to antagonists, partial agonists may cause fewer side effects since they maintain some dopaminergic tone and may be less disruptive to normal neuronal functions. We reported three sets of 4-phenylpiperazine stereoisomers that differ considerably in efficacy: the (R)-enantiomers are antagonists/weak partial agonists whereas the (S)-enantiomers are much more efficacious. To investigate the structural basis of partial agonism, we performed comparative microsecond-scale molecular dynamics simulations starting from the inactive state of D3R in complex with these enantiomers. Analysis of the simulation results reveals common structural rearrangements induced by the bound (S)-enantiomers, but not by the (R)-enantiomers, that are features of partially activated receptor conformations. These receptor models in the partial agonist-stabilized state may be useful for structure-based design of compounds with tailored efficacy profiles. Dopamine transporter DAT belongs to the Neurotransmitter:Sodium Symporter (NSS) family, and serves to terminate dopamine neurotransmission by recycling released dopamine back into the presynaptic neuron using the electro-chemical energy from the transmembrane Na+ gradient. DAT is the primary molecular target for abused psychostimulants such as cocaine and methamphetamine. Based on a wealth of information regarding the functional properties of NSSs, the crystal structures of LeuT, a bacterial NSS, reveal a central occluded substrate binding (S1) site in close association with two Na+ binding sites (the Na1 and Na2 sites), and an extracellular vestibule that binds inhibitors. Intriguingly, the configurations of these binding sites are significantly altered in various states. While these insights are critical, an understanding of the full spectrum of functional states and their transitions in a transporter cycle is required to understand the complexity of the binding modes and effects of ligands. Questions that have critical therapeutic implications are yet to be answered where and in which functional state the inhibitors bind and what their impact is on transport dynamics. The varied inhibition mechanisms of inhibitors are of particular interest in developing targeted and effective therapeutic interventions for drug abuse and other psychiatric disorders. Currently we focus on transitions among the distinct states, to reveal intermediate states to elucidate the dynamic nature of transport. Due to the lack of crystal structures of mammalian NSSs, we use the closely related LeuT and drosophila DAT as model systems to probe the mechanistic features shared within the NSS family. Two crystal structures of another prokaryotic NSS homolog, the multi-hydrophobic amino acid transporter (MhsT) from Bacillus halodurans have been resolved in novel inward-occluded states, with the extracellular vestibule closed and the intracellular portion of TM5 (TM5i) in either an unwound or a helical conformation. We have investigated the potential involvement of TM5i in Na2 binding and unbinding by carrying out comparative molecular dynamics simulations of the models derived from the two MhsT structures. We find that the helical TM5i conformation is associated with a higher propensity for Na2 release, which leads to the dissociation of the N terminus (NT) and transition to an inward-open state. By using comparative interaction network analysis, we also identify allosteric pathways connecting TM5i and the Na2 binding site to the extracellular and intracellular regions. Based on our combined computational and mutagenesis studies of MhsT and LeuT, we propose that TM5i plays a key role in Na2 binding and the conformational transition toward the inward-open state, and that the two MhsT structures represent an earlier and a later intermediate in the transport cycle that leads to full inward opening. Such a role of TM5i is likely to be shared across the NSS family. Method development Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. In order to analyze allosteric coupling networks (ACNs) in transmembrane proteins, we developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of this computational approach in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs.
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