Khelashvili George, Doktorova Milka, Sahai Michelle A, Johner Niklaus, Shi Lei, Weinstein Harel
Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York, 10065.
Proteins. 2015 May;83(5):952-69. doi: 10.1002/prot.24792. Epub 2015 Mar 25.
The dopamine transporter (DAT) is a transmembrane protein belonging to the family of neurotransmitter:sodium symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 μs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function.
多巴胺转运体（DAT）是一种跨膜蛋白，属于神经递质：钠同向转运体（NSS）家族。NSS家族成员负责从突触间隙清除神经递质，并将其转运回突触前神经末梢。DAT包含长的细胞内N端和C端结构域，这些结构域与转运体功能密切相关。特别是N端（N-term），它通过DAT调节底物的逆向转运（外流）。目前，由于缺乏N端片段的结构信息，外流的分子机制在很大程度上仍然难以捉摸。在这里，我们报告了通过从头算结构预测获得的人类DAT（hDAT）N端的计算模型，并结合脂质膜环境下广泛的原子分子动力学（MD）模拟。我们的分析表明，虽然N端是一个高度动态的结构域，但它包含在与双层相互作用的长MD轨迹中（总计>2.2微秒）保持稳定的二级结构元件。将MD模拟与连续介质平均场建模相结合，我们发现N端通过与双层中存在的带电脂质PIP2（磷脂酰肌醇4,5-二磷酸）或PS（磷脂酰丝氨酸）的静电相互作用与脂质膜结合。我们确定了N端中与这种相互作用相关的特定基序，并表明N端/膜结合的不同模式导致结构化片段在膜表面的不同定位。这些结果将为未来基于结构的研究提供信息，从而阐明N端在DAT功能中的机制作用。
