Raja M Mobeen, Kinne Rolf K H
Department of Epithelial Cell Physiology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Biochemistry. 2005 Jun 28;44(25):9123-9. doi: 10.1021/bi050323d.
We have previously shown that C-terminal loop 13 of SGLT1 acts as a major binding domain for the aglucon residues of d-glucose transport inhibitors, phlorizin (Raja, M. M., Tyagi, N. K., and Kinne, R. K. H. (2003) Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling, J. Biol. Chem. 278, 49154-49163) and alkyl glucosides (Raja, M. M., Kipp, H., and Kinne, R. K. H. (2004) C-Terminus Loop 13 of Na(+) Glucose Cotransporter SGLT1 Contains a Binding Site for Alkyl Glucosides, Biochemistry 43, 10944-10951). Topology of this loop with regard to the membrane lipids is hitherto a point of debate. Here we report on in vitro incorporation studies using fluorescence of Trp mutants of loop 13 to determine the position of various parts of the loop with the lipid bilayer. Six single Trp mutants were prepared as described in previous studies (Raja et al., 2003) and subsequently incorporated into DOPC:DOPG (60:40% molar ratio) lipid vesicles. Upon addition of the phospholipids only one mutant, R601W, exhibited no change in the fluorescence intensities, position of maxima, or acrylamide accessibility. Mutants Q581W, E621W, and L630W exhibited the most pronounced blue shifts (3-6 nm) and protection against acrylamide, suggesting a position of these segments within the lipid bilayer. This assumption was confirmed by the result that the fluorescence of only these mutants was quenched by doxyl spin membrane embedded labels in the 5- or 12-positions of the acyl side chain of phospholipids. The other parts of the peptide appear to remain outside of the lipid vesicles. Trp-591 and Trp-611 showed, although to a different extent, increase in fluorescence, blue shift of maxima, and decrease in acrylamide accessibility but no interaction with the spin-labeled phospholipids. This suggests changes in the conformation of the peptide itself. These conformation changes are probably induced by the interaction of an adjacent lysine rich region of the peptide with the negatively charged DOPG, since in the absence of this lipid no incorporation of loop 13 into the bilayer is observed. Trypsin cleavage experiments of loop 13 in proteoliposomes yield a peptide containing amino acid residues 603 to 614, confirming that this part of the loop is accessible at the extravesicular face of the membranes. The studies show that at least in the in vitro system the part of loop 13 essential for the interaction with the transport inhibitors is located extracellularly, making a similar arrangement in the intact SGLT1 probable.
我们之前已经表明，SGLT1的C末端环13是d -葡萄糖转运抑制剂根皮苷（Raja, M. M., Tyagi, N. K., and Kinne, R. K. H. (2003) Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling, J. Biol. Chem. 278, 49154 - 49163）和烷基葡糖苷（Raja, M. M., Kipp, H., and Kinne, R. K. H. (2004) C-Terminus Loop 13 of Na(+) Glucose Cotransporter SGLT1 Contains a Binding Site for Alkyl Glucosides, Biochemistry 43, 10944 - 10951）的苷元残基的主要结合结构域。迄今为止，关于该环相对于膜脂的拓扑结构一直存在争议。在此，我们报告了使用环13的色氨酸突变体的荧光进行体外掺入研究，以确定该环各部分在脂质双层中的位置。如先前研究（Raja等人，2003年）所述制备了六个单色氨酸突变体，随后将其掺入DOPC:DOPG（摩尔比60:40%）脂质囊泡中。加入磷脂后，只有一个突变体R601W的荧光强度、最大发射波长位置或丙烯酰胺可及性没有变化。突变体Q581W、E621W和L630W表现出最明显的蓝移（3 - 6纳米）以及对丙烯酰胺有保护作用，表明这些片段位于脂质双层内。仅这些突变体的荧光被嵌入磷脂酰基侧链5位或12位的多氧自由基自旋膜标记淬灭，这一结果证实了上述假设。肽的其他部分似乎留在脂质囊泡外部。Trp - 591和Trp - 611虽然程度不同，但也表现出荧光增加、最大发射波长蓝移以及丙烯酰胺可及性降低，但与自旋标记的磷脂没有相互作用。这表明肽本身的构象发生了变化。这些构象变化可能是由肽的相邻富含赖氨酸区域与带负电荷的DOPG相互作用诱导的，因为在没有这种脂质的情况下，未观察到环13掺入双层膜中。环13在蛋白脂质体中的胰蛋白酶切割实验产生了一个包含氨基酸残基603至614的肽段，证实该环的这一部分在膜的胞外表面是可及的。这些研究表明，至少在体外系统中，环13中与转运抑制剂相互作用所必需的部分位于细胞外，这使得完整的SGLT1中可能存在类似的排列。
