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，其调节的异常与神经精神和物质使用障碍有关。多巴胺D3受体(D3R)的表达因药物滥用而升高，这促使人们努力开发用于治疗药物成瘾的D3R选择性药物。抑制D3R可能不太容易引起D2R阻断所导致的运动副作用。除了抑制可卡因的行为效应外，D3R部分激动剂还可能导致更好的治疗成瘾的依从性。因此，与拮抗剂相比，部分激动剂可能引起较少的副作用，因为它们保持一定的多巴胺能张力，对正常神经功能的干扰可能较小。我们报道了三组疗效差异很大的4-苯基哌嗪立体异构体：(R)-对映体是拮抗剂/弱部分激动剂，而(S)-对映体的效果要好得多。为了研究部分激活性的结构基础，我们从D3R与这些对映体的络合物的非活性状态开始，进行了比较微秒尺度的分子动力学模拟。对模拟结果的分析揭示了结合的(S)对映体引起的常见结构重排，而不是(R)-对映体引起的结构重排，这是部分激活的受体构象的特征。这些处于部分激动剂稳定状态的受体模型可能对基于结构的化合物的设计有用，这些化合物具有定制的疗效曲线。 多巴胺转运蛋白 DAT属于神经递质：钠离子转运体(NSS)家族，通过利用跨膜Na+梯度释放的电化学能量将释放的多巴胺回收到突触前神经元中，从而终止多巴胺的神经传递。DAT是滥用可卡因和甲基苯丙胺等精神刺激剂的主要分子靶标。根据大量关于NSSS功能性质的信息，细菌NSS Leut的晶体结构揭示了一个与两个Na+结合位点(Na1和Na2位点)密切相关的中心封闭底物结合(S1)位点和一个结合抑制剂的细胞外前庭。有趣的是，这些结合位点的配置在不同的状态下发生了显著的变化。虽然这些洞察力是至关重要的，但需要了解功能状态的全谱及其在转运体周期中的转变，才能了解配体结合模式和影响的复杂性。具有关键治疗意义的问题尚未得到回答，这些抑制剂在哪里以及在哪种功能状态下结合，以及它们对运输动力学的影响是什么。抑制剂的不同抑制机制对开发针对药物滥用和其他精神疾病的有针对性和有效的治疗干预措施特别感兴趣。 目前我们关注的是不同态之间的跃迁，以揭示中间态来阐明输运的动态性质。由于哺乳动物NSSS缺乏晶体结构，我们以关系密切的Leut和果蝇DAT为模型系统，探讨NSS家族共有的机制特征。另一个原核生物NSS同系物，盐生芽孢杆菌的多疏水氨基酸转运体(MhsT)的两个晶体结构已经在新的向内封闭状态下被解析，细胞外前庭关闭，TM5(TM5i)的细胞内部分以解卷或螺旋构象存在。我们通过对两个MhsT结构得到的模型进行比较分子动力学模拟，研究了TM5i在Na2结合和解离中的潜在参与。我们发现，螺旋TM5i构象与较高的Na2释放倾向相关，这导致N末端(NT)的解离并向内开放状态转变。通过比较相互作用网络分析，我们还确定了将TM5i和Na2结合部位连接到细胞外和细胞内区域的变构通路。基于我们对MhsT和Leut的联合计算和突变研究，我们认为TM5i在Na2结合和向内开放状态的构象转变中发挥关键作用，并且这两个MhsT结构代表了导致完全向内开放的运输循环中的早期和晚期中间产物。TM5i的这种角色可能会在NSS大家庭中共享。 方法开发 跨膜蛋白参与信号处理和跨细胞膜转导的许多功能是由变构偶联决定的，变构偶联将功能效应传播到原始激活部位之外。生物化学、结晶学和单分子荧光等方面的突破为研究这类跨膜蛋白变构偶联的分子机制奠定了丰富的信息基础。然而，这些偶联的机制细节，其中许多具有治疗意义，只有在分子建模和模拟的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(ACNS)，我们开发了蛋白质相互作用分析器(PIA)，用于通过分子动力学模拟来研究结构系综中的ACNS。通过在次级活性转运蛋白和GPCRs的研究中协同进行的最新实验和计算研究的精选实例，说明了这种计算方法在询问跨膜蛋白的功能机制方面的能力。

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.