Conformational Landscape of Substrate Specificity in the Fructose Transporter GLUT5 Determined via MBAR Molecular Dynamics

Abstract

GLUT5 is a member of the glucose transporter (GLUT) family and channels fructose but not glucose through lipid membranes. Recent crystallographic data indicated that GLUT5 may possess a unique transport mechanism from other GLUT members; however, the details of this mechanism are unclear. 32μs equilibrium dynamics in combination with restrained MD were used to generate a conformational landscape of the sugar transport mechanism in four independent systems containing α-fructose, β-fructose, α-glucose, or β-glucose. A PMF was generated for each substrate using the MBAR method. The calculated PMF revealed two energy basins which were relatively consistent across the four sugars in this work. The first basin, and consequently the succeeding energy barrier, occurred at the flexible segment of the TM7 helix at the extracellular entrance of GLUT5. This flexible region was unresolved in recent GLUT5 crystal structures, highlighting its inherent flexibility. Here for the first time, we report a conformational landscape of GLUT5 and details of the required interactions of the TM7 loop with substrate. We demonstrated that initial entrance into the GLUT5 channel required overcoming an energy barrier associated with this loop. The second basin occurred in the GLUT5 central binding pocket, where the pivotal Q166 residue that is responsible for fructose specificity, made initial contact with incoming substrate. The barrier at this location was significantly higher for glucose compared to fructose. Moreover, we observed several other regions in the channel which showed specific favorable interactions with fructose. Such interactions could not occur with glucose substrates due to their minute but important steric and electrostatic incompatibilities with GLUT5. This work improved our understanding of the GLUT5 transport mechanism that is important for future development of therapies targeting sugar metabolism.