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Substrate-modulated gating dynamics in a Na~+-coupled neurotransmitter transporter homologue

机译:Na〜+耦合神经递质转运蛋白同源物的底物调控门控动力学

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摘要

Neurotransmitter/Na~+ symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na~+ electrochemical gradient~(1-6). NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine~1, as well as by antidepressants~7. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na~+ are bound deep within the protein~8. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold~(9-22). Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan~(23), and the Na~+-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy~(24). In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmem-brane helix TMla away from the transmembrane helical bundle~(22). We investigated how substrate binding is coupled to structural transitions in LeuT during Na~+-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ~10-fold faster than leucine~(15,25), we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule~(15,20). Substrate binding in this S2 site appears to act cooperatively with the primary substrate (SI) binding site to control intracellular gating more than 30 A away, in a manner that allows the Na~+ gradient to power the transport mechanism.
机译:神经递质/ Na〜+转运蛋白(NSSs)通过以Na〜+电化学梯度〜(1-6)驱动的共转运(共转运)机制重新捕获释放到突触中的神经递质来终止神经元信号传导。多巴胺,去甲肾上腺素和5-羟色胺的NSS受到精神刺激药可卡因和苯丙胺〜1以及抗抑郁药〜7的攻击。 LeuT的晶体结构是原核的NSS同源物,显示出一种封闭的构象,其中亮氨酸(Leu)和两个Na〜+结合在蛋白质〜8的深处。该结构为深入研究具有LeuT样折叠的蛋白质的功能机制奠定了基础(9-22)。随后,在抑制剂色氨酸(23)的存在下确定了“向外开放”构象,并使用电子顺磁共振波谱(24)鉴定了Na +依赖性的动态向外中间体的形成。此外,单分子荧光共振能量转移成像已被用来揭示向内开放的LeuT构象的可逆转变,这涉及跨膜螺旋TMla从跨膜螺旋束的运动[22]。我们研究了底物结合是如何在Na +耦合运输过程中耦合到LeuT中的结构转变的。在这里,我们报道了一个过程,通过该过程,来自LeuT的细胞外侧的底物结合可促进细胞内门的打开和蛋白质在细胞内表面的底物释放。在存在丙氨酸的情况下,该基质的运输速度比亮氨酸快约10倍(15,25),我们观察到丙氨酸诱导的LeuT细胞内门区域的动力学与运输效率直接相关。总的来说,我们的数据揭示了NSS转运机制的功能相关和先前隐藏的方面,这些方面强调了细胞外前庭(15,20)中第二个底物(S2)结合位点的功能重要性。在该S2位点中的底物结合似乎与主要底物(SI)结合位点协同作用,以控制Na +梯度为转运机制提供动力的方式控制了超过30 A的细胞内门控。

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  • 来源
    《Nature》 |2011年第7349期|p.109-113|共5页
  • 作者单位

    Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Department of Psychiatry, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Division of Molecular Therapeutics, New York State Psychiatric Institute, 1051 Riverside Drive, New York, New York 10032, USA;

    Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA;

    Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA,HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine,Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10021, USA;

    Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Department of Psychiatry, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Division of Molecular Therapeutics, New York State Psychiatric Institute, 1051 Riverside Drive, New York, New York 10032, USA;

    Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA,HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine,Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10021, USA;

    Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA;

    Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Department of Psychiatry, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Department of Pharmacology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA,Division of Molecular Therapeutics, New York State Psychiatric Institute, 1051 Riverside Drive, New York, New York 10032, USA;

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