How glucose transporter structure affects its function

描述（申请人提供）：葡萄糖转运蛋白（glucose transport proteins，GLUT）是一类催化平衡的细胞内糖转运的蛋白质，具有12个跨膜α螺旋（transmembrane alpha helices，TM），形成糖转运途径（两亲性TM 1、2、4、5、7、8、10和11）通过支架（疏水性TM 3、6、9和12）稳定，并采用3种构象状态：向内、封闭和向外取向。我们对GLUT理解的差距是：1）底物如何结合; 2）底物如何转运; 3）GLUT如何寡聚化。我们解决这些差距，并询问每个过程是如何改变人类疾病-GLUT 1缺乏综合征（GLUT 1-DS）。特定目的1检验底物结合GLUT易位途径的假设。GLUT 1转运葡萄糖而不是果糖，而GLUT 5转运果糖而不是葡萄糖。诱变研究表明，TM 2、7和11有助于GLUT 5果糖结合，TM 5、7、8、10和11有助于GLUT 1葡萄糖结合。使用同源性扫描诱变，我们问，如果特定的GLUT 5易位TM到GLUT 1的取代将GLUT 1从葡萄糖转换为果糖转运蛋白，反之亦然，我们问哪些残基是关键的结合。纯化的人GLUT 1的质谱分析将揭示哪些GLUT 1残基被光亲和底物/拮抗剂修饰，哪些被底物保护以防止氢氘交换（H/DX）？然后，我们询问GLUT 1-DS中的关键底物结合残基是否发生改变。具体目标2测试转运涉及由支架TM 6-途径TM 1相互作用控制的易位途径构象变化的假设。GLUT 1氢（80%）与溶剂氚或氘进行构象敏感性交换，但氨基酸交换未知。我们将通过H/DX-质谱法对向内、闭塞和向外方向捕获的纯化人GLUT 1进行鉴定。GLUT 1-GLUT 4同源性扫描突变显示，TM 6通过与TM 1相互作用控制GLUT 1和GLUT 4向内和向外方向之间的构象变化速率。我们询问这是否是所有GLUT的标志，我们通过色氨酸扫描和双半胱氨酸交联诱变研究哪些TM 6残基与TM 1相互作用，并询问这些残基是否在GLUT 1-DS中发生改变。特定目的3检验了GLUT二聚化和四聚化决定转运效率并由特定易位和支架TM介导的假设。GLUT 1形成同源二聚体和同源四聚体，而GLUT 2-4形成同源二聚体。GLUT不发生异源寡聚化。将GLUT 1支架TM 9取代到GLUT 3中允许GLUT 3四聚化并与GLUT 1形成异源复合物。GLUT 3 TM 9导致GLUT 1解离成二聚体。途径TM 5、8、2和11可能负责同种型特异性二聚化。我们建议直接测试这一假设，并概述实验，以研究GLUT活性如何受到寡聚状态的影响，以及导致GLUT 1-DS的突变是否也会影响GLUT 1四级结构和/或四级结构对功能的影响。

DESCRIPTION (provided by applicant): Glucose transport proteins (GLUTs) catalyze equilibrative, cellular sugar transport, are essential for carbohydrate homeostasis in humans, contain 12 membrane-spanning alpha helices (TMs) which form a translocation pathway (amphipathic TMs 1, 2, 4, 5, 7, 8, 10 and 11) stabilized by a scaffold (hydrophobic TMs 3, 6, 9 and 12) and adopt 3 conformational states: the inward, the occluded and the outward orientations. Gaps in our understanding of the GLUTs are: 1) How substrates bind; 2) How substrates are translocated; 3) How GLUTs oligomerize. We address these gaps and ask how each process is altered in a human disease - GLUT1 deficiency syndrome (GLUT1-DS). Specific Aim 1 tests the hypothesis that substrates bind in the GLUT translocation pathway. GLUT1 transports glucose but not fructose while GLUT5 transports fructose not glucose. Mutagenesis studies suggest that TMs 2, 7 and 11 contribute to GLUT5 fructose binding and TMs 5, 7, 8, 10 and 11 to GLUT1 glucose binding. Using homology scanning mutagenesis, we ask if substitution of specific GLUT5 translocation TMs into GLUT1 converts GLUT1 from a glucose to a fructose transporter and vice versa and we ask which residues are critical for binding. Mass spectrometry of purified human GLUT1 will reveal which GLUT1 residues are modified by photo-affinity substrates/antagonists and which are substrate- protected against hydrogen-deuterium exchange (H/DX)? We then ask if critical substrate binding residues are altered in GLUT1-DS. Specific Aim 2 tests the hypothesis that transport involves translocation pathway conformational changes controlled by scaffold TM6 - pathway TM1 interactions. GLUT1 hydrogens (80%) undergo conformation-sensitive exchange with solvent tritium or deuterium but which amino acids exchange is unknown. We will identify them by H/DX-mass spectrometry of purified human GLUT1 trapped in inward, occluded and outward orientations. GLUT1-GLUT4 homology scanning mutagenesis shows that TM6 controls the rate of conformational change between inward and outward GLUT1 and GLUT4 orientations by interacting with TM1. We ask if this is a hallmark of all GLUTs, we investigate which TM6 residues interact with TM1 by tryptophan scanning and dicysteine cross-linking mutagenesis and ask if these residues are altered in GLUT1-DS. Specific Aim 3 tests the hypothesis that GLUT dimerization and tetramerization determine transport efficiency and are mediated by specific translocation and scaffold TMs. GLUT1 forms homo- dimers and homo-tetramers whereas GLUTs 2-4 form homo-dimers. The GLUTs do not hetero-oligomerize. Substituting GLUT1 scaffold TM9 into GLUT3 allows GLUT3 to tetramerize and to form heterocomplexes with GLUT1. GLUT3 TM9 causes GLUT1 to dissociate into dimers. Pathway TMs 5, 8, 2 and 11 may be responsible for isoform specific dimerization. We propose to test this hypothesis directly and outline experiments to investigate how GLUT activity is affected by oligomeric state and whether mutations that cause GLUT1-DS also affect GLUT1 quaternary structure and/or the effects of quaternary structure on function.