Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling

M. Mobeen Raja, Navneet K. Tyagi, Rolf K. H. Kinne
2003 Journal of Biological Chemistry  
SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by sitedirected single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564 -638) were expressed in Escherichia coli and purified to homogeneity. Changes in Trp quenching and positions of the emission
more » ... a were determined after addition of phlorizin. D611W displayed the largest quenching of 80%, followed by R601W (67%). It also exhibited the maximum red shift in Trp fluorescence (ϳ14 nm), indicating an exposure of this region to a more hydrophilic environment. Titration experiments performed for each mutant showed a similar affinity for all mutants, except for D611W, which exhibited a significantly lower affinity (K d Ϸ 54 M). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (K SV ‫؍‬ 11 M ؊1 in the absence of phlorizin and 55 M ؊1 in its presence). Similar results were obtained with phloretin. CD measurements and computer modeling revealed that D611W is positioned in a random coil situated between two ␣-helical segments. By combining gel electrophoresis, enzymatic fragmentation, and matrix-assisted laser desorption ionization mass spectrometry, we also analyzed truncated loop 13 photolabeled with 3-azidophlorizin. The attachment site of the ortho-position of aromatic ring B of phlorizin was localized to Arg-602. Taken together, these data indicate that phlorizin binding elicits changes in conformation leading to a less ordered state of loop 13. Modeling suggests an interaction of the 4-and 6-OH groups of aromatic ring A of phlorizin with the region between amino acids 606 and 611 and an interaction of ring B at or around amino acid 602. Phloretin seems to interact with the same region of the protein.
doi:10.1074/jbc.m306881200 pmid:12954647 fatcat:rjyfwtmbjnbxragmjimjgsy67q