Centre National de la Recherche Scientifique, UMR 8601, Université Paris Descartes, Sorbonne Paris Cité, 45 rue des Saints-Pères, F-75006 Paris, France.
J Biol Chem. 2012 Mar 30;287(14):11489-97. doi: 10.1074/jbc.M111.313056. Epub 2012 Feb 13.
Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.
SLC17 蛋白家族的次级主动转运蛋白对于兴奋性和嘌呤能突触传递、唾液酸代谢和肾功能是必需的，其中一些成员与遗传性神经或代谢疾病有关。然而，用于研究其功能或纠正其遗传缺陷的分子工具有限或不存在。通过结构-活性、同源建模、分子对接和突变研究，我们定位了唾液酸转运蛋白（SLC17A5）的底物结合位点，该蛋白也是溶酶体唾液酸的外排体，最近也与天冬氨酸的胞吐释放有关。人类唾液酸转运蛋白在两种遗传性唾液酸储存疾病中存在缺陷，并且负责饮食中非人类唾液酸 N-羟乙酰神经氨酸的代谢掺入。我们基于与大肠杆菌甘油-3-磷酸和果糖转运蛋白的微弱但显著的序列相似性，分别构建了细胞质开放和腔开放的三维唾液酸转运蛋白模型。对这两种模型的 31 种合成唾液酸类似物的分子对接与抑制研究一致。通过两个苯丙氨酸到酪氨酸突变缩小细胞质开放状态下的唾液酸结合位点，可消除对最活跃类似物的识别，而不损害神经氨酸的转运。此外，细胞质开放模型的虚拟高通量筛选可以识别出一种假肽竞争性抑制剂，其与天然底物的亲和力高出 100 倍以上。这种经过验证的人类唾液酸转运蛋白模型和其他 SLC17 转运蛋白的唾液酸引导模型，应该为这些蛋白质的抑制剂、糖基工程工具、药理学伴侣和荧光假神经递质的鉴定铺平道路。
