Probing the Periplasmic-Open State of Lactose Permease in Response to Sugar Binding and Proton Translocation (original) (raw)
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Sugar Transport across Lactose Permease Probed by Steered Molecular Dynamics
Biophysical Journal, 2007
Escherichia coli lactose permease (LacY) transports sugar across the inner membrane of the bacterium using the proton motive force to accumulate sugar in the cytosol. We have probed lactose conduction across LacY using steered molecular dynamics, permitting us to follow molecular and energetic details of lactose interaction with the lumen of LacY during its permeation. Lactose induces a widening of the narrowest parts of the channel during permeation, the widening being largest within the periplasmic half-channel. During permeation, the water-filled lumen of LacY only partially hydrates lactose, forcing it to interact with channel lining residues. Lactose forms a multitude of direct sugar-channel hydrogen bonds, predominantly with residues of the flexible N-domain, which is known to contribute a major part of LacY's affinity for lactose. In the periplasmic halfchannel lactose predominantly interacts with hydrophobic channel lining residues, whereas in the cytoplasmic half-channel key protein-substrate interactions are mediated by ionic residues. A major energy barrier against transport is found within a tight segment of the periplasmic half-channel where sugar hydration is minimal and protein-sugar interaction maximal. Upon unbinding from the binding pocket, lactose undergoes a rotation to permeate either half-channel with its long axis aligned parallel to the channel axis. The results hint at the possibility of a transport mechanism, in which lactose permeates LacY through a narrow periplasmic half-channel and a wide cytoplasmic half-channel, the opening of which is controlled by changes in protonation states of key protein side groups.
Sugar Binding and Protein Conformational Changes in Lactose Permease
Biophysical Journal, 2006
Lactose permease is an integral membrane protein that uses the cell membrane's proton gradient for import of lactose. Based on extensive biochemical data and a substrate-bound crystal structure, intermediates involved in lactose/H 1 cotransport have been suggested. Yet, the transport mechanism, especially the coupling of protonation states of essential residues and protein conformational changes involved in the transport, is not understood. Here we report molecular-dynamics simulations of membrane-embedded lactose permease in different protonation states, both in the presence and in the absence of lactose. The results analyzed in terms of pore diameter, salt-bridge formation, and substrate motion, strongly implicate Glu 269 as one of the main proton translocation sites, whose protonation state controls several key steps of the transport process. A critical ion pair (Glu 269 and Arg 144 ) was found to keep the cytoplasmic entrance open, but via a different mechanism than the currently accepted model. After protonation of Glu 269 , the salt bridge between Glu 269 and Arg 144 was found to break, and Arg 144 to move away from Glu 269 , establishing a new salt bridge with Glu 126 ; furthermore, neutralization of Glu 269 and the displacement of Arg 144 and consequently of water molecules from the interdomain region was seen to initiate the closing of the cytoplasmic half channel (2.6-4.0 Å reduction in diameter in the cytoplasmic constriction region in 10 ns) by allowing hydrophobic surfaces of the N-and C-domains to fuse. Charged Glu 269 was found to strongly bind the lactose permeant, indicating that proton transfer from water or another residue to Glu 269 is a prerequisite for unbinding of lactose from the binding pocket.
Sugar Binding in Lactose Permease: Anomeric State of a Disaccharide Influences Binding Structure
Journal of Molecular Biology, 2007
Lactose permease in E. coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of twelve MD simulations of 20-25ns each with β(α)-D-galactopyranosyl-(1,1)-β-Dgalactopyranoside (ββ-(Galp) 2 ) and αβ-(Galp) 2 result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu-126 and αβ-(Galp) 2 has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe-118, Asn-119, Asn-240, His-322, . Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp) 2 than ββ-(Galp) 2 , i.e., −10.7±0.7 and −3.1±1.0 kcal/mol, respectively. Of the twelve helices in LacY, Helix-IV is the least stable with ββ-(Galp) 2 binding resulting in larger distortion than αβ-(Galp) 2 .
Apo-intermediate in the transport cycle of lactose permease (LacY)
Proceedings of the National Academy of Sciences, 2012
The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H + . Crystal structures reveal 12, mostly irregular, transmembrane α-helices surrounding a cavity with sugar-and H + -binding sites at the apex, which is accessible from the cytoplasm and sealed on the periplasmic side (an inward-facing conformer). An outward-facing model has also been proposed based on biochemical and spectroscopic measurements, as well as the X-ray structure of a related symporter. Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism. Here, we generate a model for an apo-intermediate of LacY based on crystallographic coordinates of LacY and the oligopeptide/H + symporter. The model exhibits a conformation with an occluded cavity inaccessible from either side of the membrane. Furthermore, kinetic considerations and double electron-electron resonance measurements suggest that another occluded conformer with bound sugar exists during turnover. An energy profile for symport is also presented. membrane proteins | modeling | membrane transport | conformational change T he major facilitator superfamily (MFS) of membrane transport proteins represents the largest family of secondary transporters, with members from Archaea to Homo sapiens (1, 2). MFS proteins catalyze transport of a wide range of substrates, including amines, acids, amino acids, sugars, peptides, and antibiotics, in many instances, by transducing the energy stored in an H + electrochemical gradient into a concentration gradient of substrate. The lactose permease of Escherichia coli (LacY), which catalyzes the coupled transport of a galactopyranoside and an H + (galactoside/H + symport) (3-6), is arguably the most extensively studied member of the MFS (7, 8). Essentially all 417 amino acyl side chains in LacY have been mutagenized (9), and functional analyses of the mutants reveals that fewer than 10 side chains play a central role in the symport mechanism ( : Glu126 (helix IV), Arg144 (helix V), and Trp151 (helix V) are directly involved in galactoside recognition and binding; Tyr236 (helix VII), Glu269 (helix VIII), and His322 (helix X) are involved in both H + translocation and affinity for sugar; and Arg302 (helix IX) and Glu325 (helix X) play important roles in H + translocation (7, 10). In the available inward-facing crystal structures of LacY (11-14), these residues are located at the apex of a deep central hydrophilic cavity, which is open to the cytoplasm only ( . The cavity is formed by 12 mostly irregular transmembrane helices organized in two pseudosymmetrical 6-helix bundles (helices I-VI and helices VII-XII) .
A Revised Model for the Structure and Function of the Lactose Permease
Journal of Biological Chemistry, 2000
The lactose permease is an integral membrane protein that cotransports H ؉ and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting the K m values (Jessen-Marshall, A. E.,
Structural determination of wild-type lactose permease
Proceedings of the National Academy of Sciences, 2007
Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H ؉ binding. To initiate transport, binding of sugar and/or an H ؉ electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.
Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY
The EMBO journal, 2006
Cation-coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H+ translocation. No sugar-binding site is observed in the absence of ligand, and deprotonation of the key residue Glu269 is associated with ligand binding. Thus, substrate induces formation of the sugar-binding site, as well as the initial step in H+ transduction.
Stochastic steps in secondary active sugar transport
Proceedings of the National Academy of Sciences of the United States of America, 2016
Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-faci...
Proceedings of the National Academy of Sciences, 2011
Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.