Structural basis for the functions of dopamine receptors and transporter

多巴胺受体 多巴胺在认知、情绪和行为功能的调节中起着重要作用，其调节异常与神经精神和物质使用障碍有关。多巴胺D3受体（D3 R）表达响应于药物滥用而升高，这促使人们努力开发用于治疗药物成瘾的D3 R选择性试剂。D3 R的抑制可能不太容易引起可能由D2 R阻断引起的运动副作用。除了抑制可卡因的行为效应外，D3 R部分激动剂可能导致更好的治疗成瘾的依从性。因此，与拮抗剂相比，部分激动剂可引起较少的副作用，因为它们维持一些多巴胺能张力，并且对正常神经元功能的破坏性较小。我们报道了三组4-苯基哌嗪立体异构体，它们在功效上有很大差异：（R）-对映体是拮抗剂/弱部分激动剂，而（S）-对映体更有效。为了研究部分激动作用的结构基础，我们进行了比较微秒尺度的分子动力学模拟，从非活性状态的D3 R与这些对映体的复合物开始。模拟结果的分析揭示了由结合的（S）-对映体诱导的常见结构重排，但不是由（R）-对映体诱导的，这是部分活化的受体构象的特征。这些受体模型在部分激动剂稳定的状态可能是有用的化合物的结构为基础的设计与定制的疗效概况。 多巴胺转运体 DAT属于神经递质：钠同向转运体（NSS）家族，并且用于通过使用来自跨膜Na+梯度的电化学能量将释放的多巴胺再循环回到突触前神经元中来终止多巴胺神经传递。DAT是滥用精神兴奋剂如可卡因和甲基苯丙胺的主要分子靶点。基于大量关于NSS功能特性的信息，细菌NSS的LeuT的晶体结构揭示了与两个Na+结合位点（Na 1和Na 2位点）密切相关的中央闭塞底物结合（S1）位点，以及结合抑制剂的细胞外前庭。有趣的是，这些结合位点的构型在不同的状态下会发生显著的变化。虽然这些见解是至关重要的，但需要了解转运体循环中功能状态及其转换的全谱，以了解配体结合模式和作用的复杂性。具有关键治疗意义的问题尚未得到回答，在哪里和在哪个功能状态的抑制剂结合，以及它们的影响是什么运输动力学。抑制剂的不同抑制机制在开发针对药物滥用和其他精神障碍的靶向和有效的治疗干预方面特别令人感兴趣。 目前，我们专注于不同的状态之间的转换，揭示中间状态，阐明运输的动态性质。由于缺乏哺乳动物NSS的晶体结构，我们使用密切相关的LeuT和果蝇DAT作为模型系统来探测NSS家族内共享的机制特征。另一个原核NSS同系物，多疏水性氨基酸转运蛋白（MhsT）从耐盐芽孢杆菌的两个晶体结构已被解决在新的向内闭塞状态，与细胞外前庭关闭和细胞内部分的TM 5（TM 5i）在解绕或螺旋构象。我们已经调查了潜在的参与TM 5i在钠结合和不结合进行比较分子动力学模拟的模型来自两个MHST结构。我们发现，螺旋TM 5i构象与较高的Na 2释放倾向，这导致N端（NT）的解离和过渡到向内开放的状态。通过使用比较相互作用网络分析，我们还确定了连接TM 5i和Na 2结合位点到细胞外和细胞内区域的变构途径。基于我们结合计算和突变的MhsT和LeuT的研究，我们提出，TM 5i起着关键作用，在钠结合和构象转变向向内开放的状态，这两个MhsT结构代表了早期和后期的中间运输周期，导致完全向内开放。TM 5i的这种作用可能在NSS家族中共享。 方法开发 跨膜蛋白的许多功能涉及跨细胞膜的信号处理和转导，这些功能是由变构偶联决定的，所述变构偶联将功能效应传播到远超过原始激活位点。从生物化学、晶体学和单分子荧光的突破中收集的数据为研究这种跨膜蛋白的变构偶联的分子机制建立了丰富的信息基础。然而，这些耦合的机制细节，其中许多具有治疗意义，只有在与分子建模和模拟协同作用下才能获得。为了分析跨膜蛋白中的变构偶联网络，我们开发了蛋白质相互作用分析仪（PIA），旨在通过分子动力学模拟来研究结构系综中的变构偶联网络。这种计算方法在询问跨膜蛋白的功能机制的权力与最近的实验和计算研究的协同进行的二级主动转运蛋白和GPCR的调查中选择的例子说明。

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.