Dakal Tikam Chand, Kumar Rajender, Ramotar Dindial
Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal, Department of Medicine, 5415 Boul. de L' Assomption, Montréal, Québec H1T 2M4, Canada.
Architecture et Fonction des Macromolécules Biologiques (AFMB), Campus de Luminy, Aix-Marseille Université, Marseille, France; Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, 160 062, Punjab, India.
Comput Biol Chem. 2017 Jun;68:153-163. doi: 10.1016/j.compbiolchem.2017.03.007. Epub 2017 Mar 18.
Human organic cation transporters (hOCTs) belong to solute carriers (SLC) 22 family of membrane proteins that play a central role in transportation of chemotherapeutic drugs for several clinical and pathological conditions, including cancer and diabetes. These transporters mediate drug transport; however, the precise mechanism of drug-binding and transport by them is not fully uncovered yet, partly due to unavailability of any crystal structure record. In this work, we performed a multi-phasic approach to compute the 3D structural models of seven human organic cation transporters (hOCTs) starting from primary protein sequence. Our structure modeling approach included 1) I-TASSER based comparative sequence alignment, threading and ab-initio protein modeling; 2) models comparison with PSIPRED secondary structure prediction; 3) loop modeling for incongruent secondary structure in Chimera 1.10.1; 4) high resolution structure simulation, refinement, energy minimization using ModRefiner, and 5) validation of the structure models using PROCHECK at SAVEs. From structural point, the computed 3D structures of hOCTs consist of a typical major facilitator superfamily (MFS) fold of twelve α-transmembrane helix domains arranged in a manner rendering hOCTs a barrel shaped structure with a large cleft that opens in cytoplasm. The modeled 3D structure of all hOCTs closely resemble to human SLC2A3 (GLUT3) transporter (PDB ID: 5c65) and displayed an outward-open confirmation and putative cyclic C1 protein symmetry. In addition, hOCTs has a large (>100 amino acids) unique extracellular loop between TMH1 and TMH2 having potential glycosylation sites (Asn-Xaa-Ser/Thr) and cysteine residues, both features indicative of putative role in drug binding and uptake. There is an intracellular three/four-helix loop between TMH6 and TMH7 containing putative phosphorylation sites for precise regulation of hOCTs function as drug transporters. There are nine loops of 4 to 11 amino acids length that protrude from membrane, both intracellularly and extracellularly, and connect adjacent TMHs. The 2D structure prediction showed N-C topology of all hOCTs. In the unavailability of the crystal structures of hOCTs, the 3D structural models computed in-silico and presented herein can be used for studying the mechanism of drug binding and transport by hOCTs.
人类有机阳离子转运体（hOCTs）属于溶质载体（SLC）22膜蛋白家族，在多种临床和病理状况（包括癌症和糖尿病）下化疗药物的转运中发挥核心作用。这些转运体介导药物转运；然而，它们药物结合和转运的确切机制尚未完全揭示，部分原因是没有任何晶体结构记录。在这项工作中，我们从蛋白质一级序列出发，采用多阶段方法计算了七种人类有机阳离子转运体（hOCTs）的三维结构模型。我们的结构建模方法包括：1）基于I-TASSER的比较序列比对、穿线法和从头算蛋白质建模；2）与PSIPRED二级结构预测进行模型比较；3）在Chimera 1.10.1中对不一致的二级结构进行环建模；4）使用ModRefiner进行高分辨率结构模拟、优化和能量最小化；5）在SAVEs中使用PROCHECK对结构模型进行验证。从结构角度来看，计算得到的hOCTs三维结构由十二个α跨膜螺旋结构域组成的典型主要转运子超家族（MFS）折叠构成，其排列方式使hOCTs呈桶状结构，在细胞质中有一个大裂缝。所有hOCTs的三维结构模型与人类SLC2A3（GLUT3）转运体（PDB ID：5c65）非常相似，呈现向外开放的构象和假定的环状C1蛋白对称性。此外，hOCTs在TMH1和TMH2之间有一个大的（>100个氨基酸）独特细胞外环，具有潜在的糖基化位点（Asn-Xaa-Ser/Thr）和半胱氨酸残基，这两个特征表明其在药物结合和摄取中可能发挥作用。在TMH6和TMH7之间有一个细胞内三/四螺旋环，含有假定的磷酸化位点，用于精确调节hOCTs作为药物转运体的功能。有九个长度为4至11个氨基酸的环从膜内和膜外突出，连接相邻的跨膜螺旋。二维结构预测显示所有hOCTs的N-C拓扑结构。在缺乏hOCTs晶体结构的情况下，本文通过计算机模拟计算得到的三维结构模型可用于研究hOCTs的药物结合和转运机制。
