(205f) Determining The Outward-Facing Structure And Sugar Binding In Lactose Permease Of e. Coli

Klauda, J., University of Maryland
Brooks, B. R., National Heart, Lung, and Blood Institute

Membrane transport proteins play significant roles in human physiology, drug transport, bacterial resistance to antibiotics, and diseases, such as cystic fibrosis. The major facilitator superfamily (MFS) is a family of membrane transport proteins found in a broad range of organisms from archaea to the human central nervous system. Lactose permease in E. coli (LacY) transports disaccharides and is studied as an important model for the MFS. The objective of our research is to determine the binding site structure and affinity of disaccharides in LacY, as well as, global structural changes from the known inward-facing to the outward-facing structure. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions by investigating LacY embedded in a fully hydrated lipid bilayer. MD simulations with galactopyranosyl-(1,1)-galactopyranoside (Galp-(1,1)-Galp) and galactopyranosyl-(1,1)-gluctopyranoside (Galp-(1,1)-Glcp) result in binding structures the depend on the anomeric state and ring structure. The Galp ring strongly interacts with Glu126, where as Asp237 and Asp240 have a greater affinity to Glcp. Significant protein-sugar interaction are seen that involve protein residues not observed in the crystal structure, as well as, those involved in proton translocation (a precursor to protein structural changes). Common to nearly all protein-sugar interactions, water acts as a hydrogen bond bridge between the disaccharide and protein. The outward-facing structure of LacY has not been determined experimentally, but opening of protein towards the periplasm is observed in our molecular simulations. Structural changes in LacY are believed to be the result of proton translocation between certain residues. Therefore, wild-type LacY is simulated with different protonated states in conjunction with normal mode analysis to obtain large-scale motions. Additional simulations of mutants that stabilize the outward-facing conformation (K131C and I129C/K131C) also verify structural transitions between the two states of the protein. These structural studies enhance our understanding of the sugar transport cycle in LacY and lay groundwork for future studies of MFS proteins.