Abstract
Although β-glucose is more abundant than α-glucose in aqueous solution, GLUT3 preferentially binds α-glucose due to favorable interactions and conformational complementarity within the protein binding site. This study explores the anomeric preferences of glucose transporters GLUT1 and GLUT3 for α- and β-glucose using classical MD, providing mechanistic insight into previously reported differences in anomer-specific transport rates during net influx, efflux, and exchange flux, as well as asymmetric binding of glucose anomer derivatives. Analysis of hydrogen-bonding frequencies between glucose anomers and transporter residues, combined with root mean squared fluctuations (RMSF) of these residues during flooding simulations, using either mixed α/β-glucose trajectories or single-anomer trajectories, reveals distinct residue preferences along the transport pathway. GLUT3 residues exposed to the extracellular solution preferentially interact with α-d-glucose, while inward-facing residues show a bias toward β-d-glucose. This distributed network of anomer-selective interactions, particularly concentrated in extramembranous surface regions, highlights previously unrecognized complexity in GLUT stereoselectivity. Enhanced residue displacements adjacent to orthosteric glucose collision sites suggest that allosteric intra- and interchain interactions may contribute to the cooperative transport behavior observed in mixed α + β-glucose simulations compared to single-anomer conditions. Importantly, anomeric stereoselectivity in GLUT1 and GLUT3 is not confined to the central high-affinity binding site, but also involves a multiplicity of extramembranous residues, underscoring the broader structural basis for selective glucose transport.