Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY (original) (raw)
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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.
Journal of Molecular Biology, 2006
Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB. Moreover, despite only 28% sequence identity and a marked difference in substrate specificity, the structural organization of the residues involved in sugar-binding and H(+) translocation is conserved in CscB. Functional analyses of mutants in the homologous key residues provide strong evidence that they play a similar critical role in the mechanisms of CscB and LacY.
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) .
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.
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.
Journal of Molecular Biology, 2010
Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics (SGLD) 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 SGLD 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), which suggest 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 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.
Journal of Molecular Biology, 2002
By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N 7 / C 5 split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-speci®c cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A Ê ), N,N H -ophenylenedimaleimide (6 A Ê ) and N,N H -p-phenylenedimaleimide (10 A Ê ). Mutant Asp240 3 Cys (helix VII)/Lys319 3 Cys (helix X) cross-links most ef®ciently with the 3 A Ê reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).
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.
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.,