How glucose transporter structure affects its function

描述(申请人提供)：葡萄糖转运蛋白(GLUT)催化平衡的细胞糖运输，对人体碳水化合物的动态平衡是必不可少的，包含12个跨膜的α螺旋(TM)，形成由支架(疏水性TM 3、6、9和12)稳定的易位途径(两亲性TM 1、2、4、5、7、8、10和11)，并采取3种构象状态：向内、被封闭和向外取向。我们对谷氨酸的理解存在以下差距：1)底物如何结合；2)底物如何转运；3)谷氨酸如何齐聚。我们解决了这些差距，并询问在一种人类疾病-GLUT1缺乏综合征(GLUT1-DS)中，每个过程是如何改变的。特定目的1检验底物结合在过剩转位途径中的假设。Glut1运输葡萄糖而不是果糖，而GLUT5运输果糖而不是葡萄糖。突变研究表明，TM 2、7和11与GLUT5果糖结合有关，TM 5、7、8、10和11与GLUT1葡萄糖结合有关。利用同源扫描突变，我们询问将特定的GLUT5易位TM替换为GLUT1是否会将GLUT1从葡萄糖转换为果糖转运体，反之亦然，我们问哪些残基对结合至关重要。纯化的人GLUT1的质谱学将揭示哪些GLUT1残基被光亲和底物/拮抗剂修饰，哪些底物受到底物保护而不受氢-氚交换(H/DX)的影响？然后我们问关键的底物结合残基在GLUT1-DS中是否发生了变化。特异性目标2验证了运输涉及转位途径构象变化的假说，该假说由支架TM6途径TM1相互作用控制。Glut1氢(80%)与溶剂氚或氚进行构象敏感的交换，但哪种氨基酸交换尚不清楚。我们将通过H/DX-MS对被困在内向、闭塞和外向的纯化的人GLUT1进行鉴定。Glut1-GLUT4同源扫描突变表明，TM6通过与TM1相互作用控制GLUT1和GLUT4向内和向外的构象变化。我们问这是否是所有GUG的标志，我们通过色氨酸扫描和二半胱氨酸交联突变调查了哪些TM6残基与TM1相互作用，并询问这些残基在GLUT1-DS中是否发生了改变。特异靶3验证了过剩二聚化和四聚化决定转运效率并由特定转位和支架TM介导的假说。Glut1形成同源二聚体和同源四聚体，而Glut2-4形成同源二聚体。谷氨酸不会异位齐聚。将GLUT1支架TM9替换到GLUT3中，可以使GLUT3四聚化并与GLUT1形成杂化络合物。GLUT3TM9使GLUT1解离成二聚体。途径TM 5、8、2和11可能与异构体特异性二聚化有关。我们建议直接检验这一假设，并概述实验，以研究寡聚态如何影响GLUT活性，以及导致GLUT1-DS的突变是否也影响GLUT1的四元结构和/或四元结构对功能的影响。

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.