Sugar binding and protein conformational changes in lactose permease

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(+) co-transport 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 A 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.     

Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.     

Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation

Hexokinase I, the pacemaker of glycolysis in brain tissue, is composed of two structurally similar halves connected by an alpha-helix. The enzyme dimerizes at elevated protein concentrations in solution and in crystal structures; however, almost all published data reflect the properties of a hexokinase I monomer in solution. Crystal structures of mutant forms of recombinant human hexokinase I, presented here, reveal the enzyme monomer for the first time. The mutant hexokinases bind both glucose 6-phosphate and glucose with high affinity to their N and C-terminal halves, and ADP, also with high affinity, to a site near the N terminus of the polypeptide chain. Exposure of the monomer crystals to ADP in the complete absence of glucose 6-phosphate reveals a second binding site for adenine nucleotides at the putative active site (C-half), with conformational changes extending 15 A to the contact interface between the N and C-halves. The structures reveal distinct conformational states for the C-half and a rigid-body rotation of the N-half, as possible elements of a structure-based mechanism for allosteric regulation of catalysis.     

Crystal structures of Entamoeba histolytica lysyl-tRNA synthetase reveal conformational changes upon lysine binding and a specific helix bundle domain

The class II lysyl-tRNA synthetases (KRS) are conserved aminoacyl-tRNA synthetases that attach lysine to the cognate tRNA in a two-step mechanism. The enzyme from the parasitic protozoan Entamoeba histolytica was crystallized in the presence of small ligands to generate snapshots of the lysine-adenylate formation. The residues involved in lysine activation are highly conserved and the active site closes around the lysyl-adenylate, as observed in bacterial KRS. The Entamoeba EMAPII-like polypeptide is not resolved in the crystals, but another Entamoeba-specific insertion could be modeled as a small helix bundle that may contribute to tRNA binding through interaction with the tRNA hinge.     

Exploration of conformational changes in lactose permease upon sugar binding and proton transfer through coarse‐grained simulations

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H⁺ symport process. Lactose/H⁺ symport is a highly complex process that involves sugar translocation, H⁺ transfer, and large‐scale protein conformational changes. The complete picture of lactose/H⁺ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse‐grained force field that couples the united‐atom protein models with the coarse‐grained MARTINI water/lipid. After validation, we implement the new force field to investigate the binding of a ‐d ‐galactopyranosyl‐1‐thio‐ ‐d ‐galactopyranoside (TDG) molecule to a wild‐type LacY. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X‐ray experiment. Transitions from inward‐facing to outward‐facing conformations upon TDG binding and protonation of Glu269 have been achieved from ～5.5 µs simulations. Both the opening of the periplasmic side and closure of the cytoplasmic side of LacY are consistent with double electron–electron resonance and thiol cross‐linking experiments. Our analysis suggests that the conformational changes of LacY are a cumulative consequence of interdomain H‐bonds breaking at the periplasmic side, interdomain salt‐bridge formation at the cytoplasmic side, and the TDG orientational changes during the transition.     

Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes

Unlike wild-type bacteriorhodopsin (BR), the BR triple mutant D96G/F171C/F219L has been shown to undergo only minor structural rearrangements during its photocycle. Nonetheless, the mutant is capable of transporting protons at a rate of 125(+/-40) H+/BR per minute under light-saturating conditions. Light adaptation of the triple mutant's retinal proceeds in a pH-dependent manner up to a maximum of 63% all-trans. These two findings imply that the transport activity of the triple mutant comprises 66% of the wild-type activity. Time-resolved spectroscopy reveals that the identity and sequence of intermediates in the photocycle of the triple mutant in the all-trans configuration correspond to that of wild-type BR. The only differences relate to a slower rise and decay of the M and O intermediates, and a significant spectral contribution from a 13-cis component. No indication for accumulation of the N intermediate is found under a variety of conditions that normally favor the formation of this species in wild-type BR. The Fourier transform infrared (FTIR) spectrum of the M intermediate in the triple mutant resembles that of wild type. Minor changes in the amide I region during the photocycle suggest that only small movements of the protein backbone occur. Electron microscopy reveals large differences in conformation between the unilluminated state of the mutant protein and wild-type but no light-induced changes in time-resolved measurements. Evidently, proton transport by the triple mutant does not require the major conformational rearrangements that occur on the same time-scale with wild-type. Thus, we conclude that large conformational changes observed in the photocycle of the wild-type and many BR mutants are not a prerequisite for the change in accessibility of the Schiff base nitrogen atom that must occur during vectorial catalysis to allow proton transport.     

Ligand-induced conformational changes in lactose repressor: a phosphorescence and ODMR study of single-tryptophan mutants.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations.     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.     

Crystal structures of a poplar thioredoxin peroxidase that exhibits the structure of glutathione peroxidases: insights into redox-driven conformational changes

Glutathione peroxidases (GPXs) are a group of enzymes that regulate the levels of reactive oxygen species in cells and tissues, and protect them against oxidative damage. Contrary to most of their counterparts in animal cells, the higher plant GPX homologues identified so far possess cysteine instead of selenocysteine in their active site. Interestingly, the plant GPXs are not dependent on glutathione but rather on thioredoxin as their in vitro electron donor. We have determined the crystal structures of the reduced and oxidized form of Populus trichocarpaxdeltoides GPX5 (PtGPX5), using a selenomethionine derivative. PtGPX5 exhibits an overall structure similar to that of the known animal GPXs. PtGPX5 crystallized in the assumed physiological dimeric form, displaying a pseudo ten-stranded beta sheet core. Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. In addition, a computer model of a complex of PtGPX5 and its in vitro recycling partner thioredoxin h1 is proposed on the basis of the crystal packing of the oxidized form enzyme. A possible role of PtGPX5 as a heavy-metal sink is also discussed.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.     

High-resolution crystal structures of Erythrina cristagalli lectin in complex with lactose and 2'-alpha-L-fucosyllactose and correlation with thermodynamic binding data

The primary sequence of Erythrina cristagalli lectin (ECL) was mapped by mass spectrometry, and the crystal structures of the lectin in complex with lactose and 2'-alpha-L-fucosyllactose were determined at 1.6A and 1.7A resolution, respectively. The two complexes were compared with the crystal structure of the closely related Erythrina corallodendron lectin (ECorL) in complex with lactose, with the crystal structure of the Ulex europaeus lectin II in complex with 2'-alpha-L-fucosyllactose, and with two modeled complexes of ECorL with 2'-alpha-L-fucosyl-N-acetyllactosamine. The molecular models are very similar to the crystal structure of ECL in complex with 2'-alpha-L-fucosyllactose with respect to the overall mode of binding, with the L-fucose fitting snugly into the cavity surrounded by Tyr106, Tyr108, Trp135 and Pro134 adjoining the primary combining site of the lectin. Marked differences were however noted between the models and the experimental structure in the network of hydrogen bonds and hydrophobic interactions holding the L-fucose in the combining site of the lectin, pointing to limitations of the modeling approach. In addition to the structural characterization of the ECL complexes, an effort was undertaken to correlate the structural data with thermodynamic data obtained from microcalorimetry, revealing the importance of the water network in the lectin combining site for carbohydrate binding.     

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 Å. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 Å and 2.6 Å, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.