Perry Jennifer L, Dembla-Rajpal Neetu, Hall Laura A, Pritchard John B
Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2006 Dec 8;281(49):38071-9. doi: 10.1074/jbc.M608834200. Epub 2006 Oct 11.
Organic anion transporters (OATs) play a critical role in the handling of endogenous and exogenous organic anions by excretory and barrier tissues. Little is known about the OAT three-dimensional structure or substrate/protein interactions involved in transport. In this investigation, a theoretical three-dimensional model was generated for human OAT1 (hOAT1) based on fold recognition to the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. GlpT and hOAT1 share several sequence motifs as major facilitator superfamily members. The structural hOAT1 model shows that helices 5, 7, 8, 10, and 11 surround an electronegative putative active site ( approximately 830A(3)). The site opens to the cytoplasm and is surrounded by three residues not previously examined for function (Tyr(230) (domain 5) and Lys(431) and Phe(438) (domain 10)). Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed by point mutations in a Xenopus oocyte expression system. Membrane protein expression was severely limited for the Y230A mutant. For the K431A and F438A mutants, [(3)H]PAH uptake was less than 30% of wild-type hOAT1 uptake after protein expression correction. Reduced V(max) values for the F438A mutant confirmed lower protein expression. In addition, the F438A mutant exhibited an increased affinity for cidofovir but was not significantly different for PAH. Differences in handling of PAH and cidofovir were also observed for the Y230F mutant. Little uptake was determined for cidofovir, whereas PAH uptake was similar to wild-type hOAT1. Therefore, the hOAT1 structural model has identified two new residues, Tyr(230) and Phe(438), which are important for substrate/protein interactions.
有机阴离子转运体（OATs）在排泄组织和屏障组织处理内源性和外源性有机阴离子的过程中发挥着关键作用。关于OAT的三维结构或转运过程中底物与蛋白质的相互作用，我们所知甚少。在本研究中，基于对大肠杆菌磷酸甘油转运体（GlpT）晶体结构的折叠识别，生成了人OAT1（hOAT1）的理论三维模型。GlpT和hOAT1作为主要易化子超家族成员，共享多个序列基序。hOAT1的结构模型显示，螺旋5、7、8、10和11围绕着一个带负电的假定活性位点（约830ų）。该位点向细胞质开放，并被三个以前未进行功能研究的残基所包围（结构域5中的Tyr²³⁰以及结构域10中的Lys⁴³¹和Phe⁴³⁸）。通过非洲爪蟾卵母细胞表达系统中的点突变，评估了这些残基对对氨基马尿酸（PAH）和西多福韦转运的影响。Y230A突变体的膜蛋白表达严重受限。对于K431A和F438A突变体，在校正蛋白质表达后，[³H]PAH摄取量不到野生型hOAT1摄取量的30%。F438A突变体的Vmax值降低，证实其蛋白质表达较低。此外，F438A突变体对西多福韦的亲和力增加，但对PAH的亲和力无显著差异。Y230F突变体在PAH和西多福韦处理上也存在差异。西多福韦的摄取量很少，而PAH摄取量与野生型hOAT1相似。因此，hOAT1结构模型确定了两个新的残基Tyr²³⁰和Phe⁴³⁸，它们对底物与蛋白质的相互作用很重要。
