Bridging the gap between structure and kinetics of human SGLT1
Bridging the gap between structure and kinetics of human SGLT1
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DOI：
10.1152/ajpcell.00397.2011
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发表时间：
2012-05-01
影响因子：
5.5
通讯作者：
Wright, Ernest M.
中科院分区：
文献类型：
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作者：
Sala-Rabanal, Monica;Hirayama, Bruce A.;Wright, Ernest M.
Sala-Rabanal M，Hirayama BA，Loo DDF，Chaptal V，Abramson J，Wright EM.弥合人SGLT 1的结构和动力学之间的差距。美国生理学杂志细胞生理学302：C1293-C1305，2012年。首次发表于2011年12月7日; doi：10.1152/ajpcell.00397.2011。Na+-葡萄糖协同转运蛋白hSGLT 1是一类利用Na+电化学梯度驱动溶质向上转运的膜蛋白的成员。虽然hSGLT 1属于一个基因家族（SLC 5），但最近对细菌Na+协同转运蛋白的结构研究表明，不同基因家族中的Na+转运蛋白具有相同的结构折叠。我们构建了hSGLT 1的两种构象的同源模型，向内封闭（基于vSGLT）和向外开放构象（基于Mhp 1），依次突变每个保守的门和配体结合残基，在非洲爪蟾卵母细胞中表达SGLT 1突变体，并使用生物物理和生化测定确定功能后果。结果证实，突变配体结合残基产生配体亲和力的深刻变化（半饱和浓度，K-0.5）;例如，在一个实施例中，突变糖结合残基使葡萄糖K-0.5增加高达三个数量级。突变的外部门残基增加Na+糖运输的化学计量，表明这些残基是关键的有效的共运输。根皮苷抑制常数（Ki）的变化与糖K-0.5的变化成比例，除了在F101 C的情况下，其中根皮苷Ki增加几个数量级而葡萄糖K-0.5没有变化。我们的结论是，葡萄糖和根皮苷占据相同的结合位点，F101参与结合的根皮素组的抑制剂。取代的半胱氨酸可及性方法表明，在门和糖结合位点的位置处的半胱氨酸残基在很大程度上仅在外部Na+的存在下可接近外部亲水性甲硫基磺酸盐试剂，表明外部糖（和根皮苷）结合前庭通过外部Na+的存在而打开，并且在糖和根皮苷结合后关闭。总体而言，本研究结果提供了动力学和结构研究的cotorpers之间的桥梁。
Sala-Rabanal M, Hirayama BA, Loo DDF, Chaptal V, Abramson J, Wright EM. Bridging the gap between structure and kinetics of human SGLT1. Am J Physiol Cell Physiol 302: C1293-C1305, 2012. First published December 7, 2011; doi:10.1152/ajpcell.00397.2011.-The Na+-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na+ electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na+ cotransporters have shown that Na+ transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K-0.5); e. g., mutating sugar binding residues increases the glucose K-0.5 by up to three orders of magnitude. Mutation of the external gate residues increases the Na+ to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K-i) are proportional to the changes in sugar K-0.5, except in the case of F101C, where phlorizin Ki increases by orders of magnitude without a change in glucose K-0.5. We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na+, demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na+ and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.