From the Department of Biochemistry and Molecular Pharmacology, Graduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
From the Department of Biochemistry and Molecular Pharmacology, Graduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester, Massachusetts 01605
J Biol Chem. 2017 Dec 22;292(51):21035-21046. doi: 10.1074/jbc.M117.815589. Epub 2017 Oct 24.
Recent structural studies suggest that GLUT1 (glucose transporter 1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple β-d-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single β-d-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the for 2-deoxy-d-glucose uptake and eliminated -allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not -allostery (uptake stimulation by subsaturating cytochalasin B). -Allostery persisted, but -allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated -allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) -allostery is mediated by intrasubunit interactions and 2) -allostery requires intersubunit interactions.
最近的结构研究表明，GLUT1（葡萄糖转运蛋白 1）介导的糖转运是由交替访问转运体介导的，该转运体依次呈现外（e2）和内（e1）底物结合位点。然而，转运研究表明存在多个相互作用（变构）和共存的外和内 GLUT1 配体结合位点。本研究旨在探讨这些相互矛盾的结论是源于系统分析错误还是揭示了转运体结构与功能之间更基本的关系。在这里，同源建模通过确认至少四种 GLUT1 构象（所谓的外向、外向闭塞、内向闭塞和内向 GLUT1 构象），支持了糖转运的交替访问转运体模型。对接分析的结果表明，外向和外向闭塞构象呈现多个β-d-葡萄糖和麦芽糖相互作用位点，而内向闭塞和内向构象仅呈现单个β-d-葡萄糖相互作用位点。Gln-282 通过氢键参与所有 GLUT1 构象中的糖结合。将 Gln-282 突变为丙氨酸（Q282A）使 2-脱氧-d-葡萄糖摄取的 Km 增加了一倍，并消除了变构作用（通过亚饱和细胞外麦芽糖刺激糖摄取），但没有消除变构作用（通过亚饱和细胞松弛素 B 刺激摄取）。变构作用持续存在，但在我们用 GLUT3 序列取代膜螺旋 9 的寡聚化缺陷 GLUT1 变体中，变构作用丧失。此外，Q282A 在寡聚化变体中消除了变构作用。这些发现通过以下方式调和结构和转运研究中的矛盾结论：GLUT1 是变构交替访问转运体的寡聚物，其中 1）变构作用由亚基内相互作用介导，2）变构作用需要亚基间相互作用。
