Analysis of Glucose Transporter Topology and Structural Dynamics
Analysis of Glucose Transporter Topology and Structural Dynamics
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DOI：
10.1074/jbc.m804802200
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发表时间：
2008-12-26
影响因子：
4.8
通讯作者：
Carruthers, Anthony
中科院分区：
文献类型：
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作者：
Blodgett, David M.;Graybill, Christopher;Carruthers, Anthony
同源模建和扫描半胱氨酸突变研究表明，人葡萄糖转运蛋白GLUT1与其远端细菌同源物Lacy和GlpT具有相似的结构。我们通过绘制纯化的、重组的人红细胞GLUT1对水探针的可及性的图谱来检验这一假设。Glut1含有35个潜在的胰酶切割位点。16个赖氨酸残基中的14个和19个精氨酸残基中的18个可被胰酶消化。用异硫氰酸酯和N-羟基丁二酰亚胺(NHS)酯以底物依赖的方式修饰谷氨酸1赖氨酸残基。磺基-NHS-LC-生物素可与12个赖氨酸残基结合。Glut1胰酶从膜上释放出全长的跨膜螺旋1、胞质环6-7和长的胞质C末端。胰酶消化的GLUT1保留了细胞松弛素B和D-葡萄糖的结合能力，并在细胞松弛素B(但不是D-葡萄糖)结合时释放了全长的跨膜螺旋8。跨膜螺旋8的释放不能消除细胞松弛素B的结合。Glut1被α-胰凝乳蛋白酶广泛地降解，它在多个位置切割可能形成孔的两亲性α-螺旋1、2、4、7、8、10和11，将跨膜多肽片段释放到水溶液中。推测的支架膜螺旋3、6、9和12具有很强的疏水性，对α-胰凝乳蛋白酶具有抵抗力，并被膜双层所保留。这些观察结果为所提议的GLUT1结构提供了实验支持；表明所提议的膜螺旋5、6和12的拓扑需要调整；并表明GLUT1支架内跨膜螺旋1和8的亚稳构象破坏了糖转运中间体的稳定性。
Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and D-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.