title: Probing the periplasmic-open state of lactose permease in response to sugar binding and proton translocation.
abstract: Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires self-guided Langevin dynamics to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), suggesting a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation of Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42, and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA.
authors.forename: Pushkar Y
authors.initials: PY
authors.lastname: Pendse
authors.forename: Bernard R
authors.initials: BR
authors.lastname: Brooks
authors.forename: Jeffery B
authors.initials: JB
authors.lastname: Klauda
mesh_terms: D001665:Binding Sites; D050260:Carbohydrate Metabolism; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D011522:Protons; D011994:Recombinant Proteins; D027981:Symporters