Monitoring conformational rearrangements in the substrate-binding site of a membrane transport protein by mass spectrometry

Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low ( approximately 25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding.     

Sugar recognition by the lactose permease of Escherichia coli

Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.     

Aromatic stacking in the sugar binding site of the lactose permease

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.     

Sugar recognition by CscB and LacY

The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily, and both catalyze sugar/H(+) symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose, and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C(3)-OH group of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of CscB is directed toward the fructofuranosyl moiety of the substrate, while the specificity of LacY is directed toward the galactopyranosyl moiety.     

Manipulating conformational equilibria in the lactose permease of Escherichia coli.

A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized. Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed. In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored.     

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.     

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.     

beta-D-Galactoside transport in Escherichia coli: substrate recognition.

1. A number of galactosides and other sugar compounds were examined as inhibitors of facilitated or active transport by the lactose permease system of Escherichia coli. Efficient inhibition required an alpha- or beta-anomeric galactopyranosyl ring of D-configuration, a free 6-hydroxyl group, and a certain aglycone size which was reached, for example, by monosaccharide or nitrophenyl substituents. 2. Aromatic alpha-D-galactopyranosides acted as high-affinity inhibitors (Ki, below 50 micrometer). At least two of them were not transported, in contrast to alpha-galactoside disaccharides and to aromatic beta-D-galactopyranosides. 3. beta-D-Galactoside transport was not significantly inhibited by specific inhibitors and transitionstate analogues of beta-galactosidase (D-galactal, D-galactonolascone). 4. The beta-D-galactopyranoside, lactitol, and alpha-D-galactopyranoside, galactinol, were not efficiently bound by the lactose permease system, although the maximal rate of uptake of lacitol was similar to that of lactose. By comparison with several structurally related D-galactopyranosides, the decreased affinity was attributed to an effect of the membrane/water interface. A model for substrate recognition by the lactose permease system is presented.     

Functional conservation in the putative substrate binding site of the sucrose permease from Escherichia coli

The sucrose (CscB) permease is the only member of the oligosaccharide:H(+) symporter family in the Major Facilitator Superfamily that transports sucrose but not lactose or other galactosides. In lactose permease (lac permease), the most studied member of the family, three residues have been shown to participate in galactoside binding: Cys148 hydrophobically interacts with the galactosyl ring, while Glu126 and Arg144 are charge paired and form H-bonds with specific galactosyl OH groups. In the present study, the role of the corresponding residues in sucrose permease, Asp126, Arg144, and Ser148, is investigated using a functional Cys-less mutant (see preceding paper). Replacement of Ser148 with Cys has no significant effect on transport activity or expression, but transport becomes highly sensitive to the sulfhydryl reagent N-ethylmaleimide (NEM) in a manner similar to that of lac permease. However, in contrast to lac permease, substrate affords no protection whatsoever against NEM inactivation of transport or alkylation with [(14)C]NEM. Neutral (Ala, Cys) mutations of Asp126 and Arg144 abolish sucrose transport, while membrane expression is not affected. Similarly, combination of two Ala mutations within the same molecule (Asp126-->Ala/Arg144-->Ala) yields normally expressed, but completely inactive permease. Conservative replacements result in highly active molecules: Asp126-->Glu permease catalyzes sucrose transport comparable to Cys-less permease, while mutant Arg144-->Lys exhibits decreased but significant activity. The observations demonstrate that charge pair Asp126-Arg144 plays an essential role in sucrose transport and suggest that the overall architecture of the substrate binding sites is conserved between sucrose and lac permeases.     

Engineering conformational flexibility in the lactose permease of Escherichia coli: use of glycine-scanning mutagenesis to rescue mutant Glu325-->Asp

Lactose/H(+) symport by lactose permease of Escherichia coli involves interactions between four irreplaceable charged residues in transmembrane helices that play essential roles in H(+) translocation and coupling [Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Arg144 (helix V) which are obligatory for substrate binding. The conservative mutation Glu325-->Asp causes a 10-fold reduction in the V(max) for active lactose transport and markedly decreased lactose-induced H(+) influx with no effect on exchange or counterflow, neither of which involves H(+) symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325-->Asp in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325-->Asp background are identified that exhibit significantly higher transport activity. Furthermore, mutant Val316-->Gly/Glu325-->Asp catalyzes active transport, efflux, and lactose-induced H(+) influx with kinetic properties approaching those of wild-type permease. It is proposed that introduction of conformational flexibility at the interface between helices IX and X improves juxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane [Sahin-Tóth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10729-10732.     

Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with a water-soluble carbodiimide: identification of carboxyl groups protected by MgATP and inhibitor peptides

The catalytic subunit of cAMP-dependent protein kinase typically phosphorylates protein substrates containing basic amino acids preceding the phosphorylation site. To identify amino acids in the catalytic subunit that might interact with these basic residues in the protein substrate, the enzyme was treated with a water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), in the presence of [14C]glycine ethyl ester. Modification of the catalytic subunit in the absence of substrates led to the irreversible, first-order inhibition of activity. Neither MgATP nor a 6-residue inhibitor peptide alone was sufficient to protect the catalytic subunit against inactivation by the carbodiimide. However, the inhibitor peptide and MgATP together completely blocked the inhibitory effects of EDC. Several carboxyl groups in the free catalytic subunit were radiolabeled after the catalytic subunit was modified with EDC and [14C]glycine ethyl ester. After purification and sequencing, these carboxyl groups were identified as Glu 107, Glu 170, Asp 241, Asp 328, Asp 329, Glu 331, Glu 332, and Glu 333. Three of these amino acids, Glu 331, Glu 107, and Asp 241, were labeled regardless of the presence of substrates, while Glu 333 and Asp 329 were modified to a slight extent only in the free catalytic subunit. Glu 170, Asp 328, and Glu 332 were all very reactive in the apoenzyme but fully protected from modification by EDC in the presence of MgATP and an inhibitor peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

Structure-Function Studies of a Melanocarpus albomyces Laccase Suggest a Pathway for Oxidation of Phenolic Compounds

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.     

Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY

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.     

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.     

Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.

Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.     

Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli.

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity. To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5. In addition, no binding is detected with Glu126-->His or Arg144-->His permease. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity.     

Helices VII and X in the lactose permease of Escherichia coli: proximity and ligand-induced distance changes.

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-specific cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A), N,N'-o- phenylenedimaleimide (6 A) and N,N'-p-phenylenedimaleimide (10 A). Mutant Asp240-->Cys (helix VII)/Lys319-->Cys (helix X) cross-links most efficiently with the 3 A 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).     

Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA

The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site.