Palazzolo Luca, Parravicini Chiara, Laurenzi Tommaso, Guerrini Uliano, Indiveri Cesare, Gianazza Elisabetta, Eberini Ivano
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy.
Dipartimento di Biologia, Ecologia e Scienze della Terra, University of Calabria, Cosenza, Italy.
Front Chem. 2018 Aug 24;6:350. doi: 10.3389/fchem.2018.00350. eCollection 2018.
The molecular mechanism of transport mediated by LAT1, a sodium-independent antiporter of large neutral amino acids, was investigated through procedures, specifically making reference to two transported substrates, tyrosine (Tyr) and leucine methyl ester (LME), and to 3,5-diiodo-L-tyrosine (DIT), a well-known LAT1 inhibitor. Two models of the transporter were built by comparative modeling, with LAT1 either in an outward-facing (OF) or in an inward-facing (IF) conformation, based, respectively, on the crystal structure of AdiC and of GadC. As frequently classic Molecular Dynamics (MD) fails to monitor large-scale conformational transitions within a reasonable simulated time, the OF structure was equilibrated for 150 ns then processed through targeted MD (tMD). During this procedure, an elastic force pulled the OF structure to the IF structure and induced, at the same time, substrates/inhibitor to move through the transport channel. This elastic force was modulated by a spring constant () value; by decreasing its value from 100 to 70, it was possible to comparatively account for the propensity for transport of the three tested molecules. In line with our expectations, during the tMD simulations, Tyr and LME behaved as substrates, moving down the transport channel, or most of it, for all values. On the contrary, DIT behaved as an inhibitor, being (almost) transported across the channel only at the highest value (100). During their transit through the channel, Tyr and LME interacted with specific amino acids (first with Phe252 then with Thr345, Arg348, Tyr259, and Phe262); this suggests that a primary as well as a putative secondary gate may contribute to the transport of substrates. Quite on the opposite, DIT appeared to establish only transient interactions with side chains lining the external part of the transport channel. Our tMD simulations could thus efficiently discriminate between two transported substrates and one inhibitor, and therefore can be proposed as a benchmark for developing novel LAT1 inhibitors of pharmacological interest.
通过相关程序研究了LAT1（一种大型中性氨基酸的钠非依赖性反向转运体）介导的转运分子机制，特别参考了两种转运底物，即酪氨酸（Tyr）和亮氨酸甲酯（LME），以及一种著名的LAT1抑制剂3,5 - 二碘 - L - 酪氨酸（DIT）。基于AdiC和GadC的晶体结构，通过比较建模构建了两种转运体模型，分别为外向构象（OF）和内向构象（IF）的LAT1。由于经典分子动力学（MD）常常无法在合理的模拟时间内监测大规模构象转变，因此对OF结构进行了150纳秒的平衡，然后通过靶向分子动力学（tMD）进行处理。在此过程中，一个弹性力将OF结构拉向IF结构，同时诱导底物/抑制剂通过转运通道移动。该弹性力由弹簧常数（）值调节；通过将其值从100降至70，可以比较三种测试分子的转运倾向。正如我们所预期的，在tMD模拟过程中，Tyr和LME表现为底物，在所有值下都沿着转运通道或其大部分移动。相反，DIT表现为抑制剂，仅在最高值（100）时（几乎）穿过通道。在它们通过通道的过程中，Tyr和LME与特定氨基酸相互作用（首先与Phe252相互作用，然后与Thr345、Arg348、Tyr259和Phe262相互作用）；这表明一个主要以及一个假定的次要门控可能有助于底物的转运。恰恰相反，DIT似乎仅与位于转运通道外部的侧链建立短暂相互作用。因此，我们的tMD模拟可以有效地区分两种转运底物和一种抑制剂，因此可以作为开发具有药理学意义的新型LAT1抑制剂的基准。
