Humanization of the Substrate Binding Site in vSGLT

Secondary active transporters utilize the energy of electrochemical gradients to drive the uphill transport of a second substrate. Sodium Glucose Cotransporters (SGLT) constitute a large family of symporters that use Na+ ions to cotransport glucose or galactose as well as a variety of other substrates. SGLTs are responsible for the absorption of dietary sugars in the intestine and reabsorption from primary urine in the kidney. Recently, inhibitors of SGLTs came into focus as novel drugs to treat diabetes. Phlorizin-derivatives competitively inhibit reabsorption of sugar from the glomerular filtrate, thereby increasing its excretion in the urine and ultimately lowering blood sugar concentrations.However, to this date, no structural information on human SGLTs is available. Only the 3D structure for the bacterial homologue vSGLT from Vibrio parahaemolyticus which does not bind phlorizin, has been solved. Therefore the binding mechanism of these inhibitors still remains not very well understood. To gain further insights into the determinants for phlorizin binding, we designed several point mutants to mimic the presumed binding site in human SGLT. Subsequently we determined sugar and phlorizin binding to the purified mutant proteins by microscale thermophoresis. This information will increase the value of vSGLT as a model system sodium-dependent sugar transport in humans and might ultimately help to design new and more specific inhibitors.