D-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/D-Glucose Cotransporter 1 (hSGLT1): A Tryptophan Scanning Study

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• 作者：Navneet K. Tyagi ; Azad Kumar ; Pankaj Goyal ; Dharmendra Pandey ; Wolfgang Siess ; Rolf K. H. Kinne
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 27, 2007
• 年：2007
• 卷：46
• 期：47
• 页码：13616 - 13628
• 全文大小：233K
• 年卷期：v.46,no.47(November 27, 2007)
• ISSN：1520-4995

文摘

In order to gain a better understanding of the structure-function relation in hSGLT1, singleTrp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previouslyhad been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutantproteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transportexperiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drasticdecrease in affinity toward -methyl-D-glucopyranoside with K_m values of 13.3 and 5.26 mM comparedto 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin wasobserved. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivityto N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate thatTrp⁴⁵⁷ and Trp⁴⁶⁰ are in contact with the hydrophilic extravesicular environment. In both mutants Trpfluorescence was quenched significantly, but differently, by various glucose analogues. They also showsignificant protection by D-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity,and show fluorescence properties which indicate that these residues are located in a very hydrophilicenvironment. Phlorizin and phloretin, but not D-glucose, protect both mutants against collisional quenchers.Depth-calculations using the parallax method suggest a location of Trp⁴⁵⁷ and Trp⁴⁶⁰ at an average distanceof 10.8 Å and 7.4 Å from the center of the bilayer, while Trp⁶⁰² and Trp⁶⁰⁹ are located outside the membrane.These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 residein a hydrophilic access pathway extending 5-7 Å into the membrane to which sugars as well as the sugarmoiety of inhibitory glucosides bind. Residues Phe⁶⁰² and Phe⁶⁰⁹ contribute by their hydrophobic aromaticresidues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex aclose vicinity between these two subdomains of the transporter is established creating a phlorizin bindingpocket with the previously estimated dimensions of 10 × 17 × 7 Å.