Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop

GltS of Escherichia coli is a secondary transporter that catalyzes Na+-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na+ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na+ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.     

Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering

Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.     

Cryo-EM Structures of Azospirillum brasilense Glutamate Synthase in Its Oligomeric Assemblies

Bacterial NADPH-dependent glutamate synthase (GltS) is a complex iron–sulfur flavoprotein that catalyzes the reductive synthesis of two L-Glu molecules from L-Gln and 2-oxo-glutarate. GltS functional unit hosts an α-subunit (αGltS) and a β-subunit (βGltS) that assemble in different αβ oligomers in solution. Here, we present the cryo-electron microscopy structures of Azospirillum brasilense GltS in four different oligomeric states (α₄β₃, α₄β₄, α₆β₄ and α₆β₆, in the 3.5- to 4.1-Å resolution range). Our study provides a comprehensive GltS model that details the inter-protomeric assemblies and allows unequivocal location of the FAD cofactor and of two electron transfer [4Fe–4S]^+1,+2 clusters within βGltS.     

The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit

Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain.     

Structural studies on the synchronization of catalytic centers in glutamate synthase

The complex iron-sulfur flavoprotein glutamate synthase (GltS) plays a prominent role in ammonia assimilation in bacteria, yeasts, and plants. GltS catalyzes the formation of two molecules of l-glutamate from 2-oxoglutarate and l-glutamine via intramolecular channeling of ammonia. GltS has the impressive ability of synchronizing its distinct catalytic centers to avoid wasteful consumption of l-glutamine. We have determined the crystal structure of the ferredoxin-dependent GltS in several ligation and redox states. The structures reveal the crucial elements in the synchronization between the glutaminase site and the 2-iminoglutarate reduction site. The structural data combined with the catalytic properties of GltS indicate that binding of ferredoxin and 2-oxoglutarate to the FMN-binding domain of GltS induce a conformational change in the loop connecting the two catalytic centers. The rearrangement induces a shift in the catalytic elements of the amidotransferase domain, such that it becomes activated. This machinery, over a distance of more than 30 A, controls the ability of the enzyme to bind and hydrolyze the ammonia-donating substrate l-glutamine.     

Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates

Glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of L-glutamine amide group to the C2 carbon of 2-oxoglutarate yielding two molecules of L-glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS alpha subunit and the structure of the reduced enzyme in complex with the L-glutamine and 2-oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS alpha subunit in complex with L-methionine sulphone and 2-oxoglutarate. The present 4-ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263-271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q-loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L-methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid-body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS alpha subunit.     

Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn 3phoA . The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty-eight unique phoA -positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T-DNA transport pore is presented.     

Structure--function studies on the iron-sulfur flavoenzyme glutamate synthase: an unexpectedly complex self-regulated enzyme

Glutamate synthase (GltS) is, with glutamine synthetase, the key enzyme of ammonia assimilation in bacteria, microorganisms and plants. GltS isoforms result from the assembly and co-evolution of conserved functional domains. They share a common mechanism of reductive glutamine-dependent glutamate synthesis from 2-oxoglutarate, which takes place within the alpha subunit ( approximately 150 kDa) of the NADPH-dependent bacterial enzyme and the corresponding polypeptides of other GltS forms, and involves: (i) an Ntn-type amidotransferase domain and (ii) a flavin mononucleotide-containing (beta/alpha)(8) barrel synthase domain connected by (iii) a approximately 30 A-long intramolecular ammonia tunnel. The synthase domain harbors the [3Fe/4S](0,+1) cluster of the enzyme, which participates in the electron transfer process from the physiological reductant: reduced ferredoxin in the plant-type enzyme or NAD(P)H in the bacterial and the non-photosynthetic eukaryotic form. The NAD(P)H-dependent GltS requires a tightly bound flavin adenine dinucleotide-dependent reductase (beta subunit, approximately 50 kDa), also determining the presence of two low-potential [4Fe-4S](+1,+2) clusters. Structural, functional and computational data available on GltS and related enzymes show how the enzyme may control and coordinate the reactions taking place at the glutaminase and synthase sites by sensing substrate binding and cofactor redox state.     

Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry

We have established a protocol to study the kinetics of incorporation of 15N into glutamine (Gln), glutamic acid (Glu), alanine (Ala) and proline (Pro) in Aedes aegypti females. Mosquitoes were fed 3% sucrose solutions containing either 80 mM 15NH4Cl or 80 mM glutamine labeled with 15N in either the amide nitrogen or in both amide and amine nitrogens. In some experiments, specific inhibitors of glutamine synthetase or glutamate synthase were added to the feeding solutions. At different times post feeding, which varied between 0 and 96 h, the mosquitoes were immersed in liquid nitrogen and then processed. These samples plus deuterium labeled internal standards were derivatized as dimethylformamidine isobutyl esters or isobutyl esters. The quantification of 15N-labeled and unlabeled amino acids was performed by using mass spectrometry techniques. The results indicated that the rate of incorporation of 15N into amino acids was rapid and that the label first appeared in the amide side chain of Gln and then in the amino group of Gln, Glu, Ala and Pro. The addition of inhibitors of key enzymes related to the ammonia metabolism confirmed that mosquitoes efficiently metabolize ammonia through a metabolic route that mainly involves glutamine synthetase (GS) and glutamate synthase (GltS). Moreover, a complete deduced amino acid sequence for GltS of Ae. aegypti was determined. The sequence analysis revealed that mosquito glutamate synthase belongs to the category of NADH-dependent GltS.     

Cross-linking of dimeric CitS and GltS transport proteins

CitS of Klebsiella pneumoniae and GltS of Escherichia coli are Na+-dependent secondary transporters from different families that are believed to share the same fold and quaternary structure. A 10 kDa protein tag (Biotin Acceptor Domain [BAD]) was fused to the N-terminus of both proteins (CitS-BAD1 and GltS-BAD1, respectively) and inserted in the central cytoplasmic loop that connects the two halves of the proteins (CitS-BAD260 and GltS-BAD206). Both CitS constructs and GltS-BAD206 were produced and shown to be active transporters, but GltS-BAD1 could not be detected in the membrane. Distance relationships in the complexes were studied by cross-linking studies. Both CitS constructs were shown to be in the dimeric state after purification in detergent by cross-linking with glutaraldehyde. The concentration of glutaraldehyde resulting in 50% cross-linking was significantly higher for CitS-BAD1 than for CitS and CitS-BAD260. Remarkably, GltS and GltS-BAD260 were not cross-linked by glutaraldehyde because of the lack of productive reactive sites. Cross-linking of GltS was observed when the N-terminal 46 residues of CitS with or without BAD at the N-terminus were added to the N-terminus of GltS. The stretch of 46 residues contains the first transmembrane segment of CitS that is missing in the GltS structure. The data support an orientation of the monomers in the dimer with the N-termini close to the dimer interface and the central cytoplasmic loops far away at the ends of the long axis of the dimer structure in a view perpendicular to the membrane.     

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.     

Membrane topology of the NixA nickel transporter of Helicobacter pylori: two nickel transport-specific motifs within transmembrane helices II and III

NixA, the high-affinity cytoplasmic membrane nickel transport protein of Helicobacter pylori, imports Ni(2+) into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with beta-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni(2+) transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni(2+) transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.     

Topological studies on the twin-arginine translocase component TatC

The twin-arginine translocation (Tat) system can translocate folded proteins across biological membranes. Among the known Tat-system components in Escherichia coli, TatC is the only protein with multiple trans-membrane domains. TatC is important for translocon interactions with Tat substrates. The knowledge of its membrane topology is therefore crucial for the understanding of substrate binding and translocon function. Recently, based on active PhoA reporter fusions to the second predicted cytoplasmic loop of TatC, a topology with four trans-membrane domains has been suggested, calling in silico predictions of six trans-membrane domains into question. Here we report studies with translational fusions of TatC to the topological marker enzymes PhoA and LacZ which provide strong evidence for a six-trans-membrane domain topology. The stop transfer capacity of the fourth trans-membrane domain was found to be strongly influenced by the succeeding cytoplasmic domain. The presence of linker sequences at PhoA-fusion sites of the cytoplasmic domain induced PhoA leakage. In the case of one tested fusion (S185-PhoA), the stop-transfer efficiency was already low due to the negative charge in the center of the fourth trans-membrane domain (E170). The results point to the importance of cytoplasmic loops for the stabilization of stop-transfer sequences and revoke evidence for only four trans-membrane domains of TatC.     

Membrane topology prediction by hydropathy profile alignment: membrane topology of the Na(+)-glutamate transporter GltS

Structural classification of families of membrane proteins by bioinformatics techniques has become a critical aspect of membrane protein research. We have proposed hydropathy profile alignment to identify structural homology between families of membrane proteins. Here, we demonstrate experimentally that two families of secondary transporters, the ESS and 2HCT families, indeed share similar folds. Members of the two families show highly similar hydropathy profiles but cannot be shown to be homologous by sequence similarity. A structural model was predicted for the ESS family transporters based upon an existing model of the 2HCT family transporters. In the model, the transporters fold into two domains containing five transmembrane segments and a reentrant or pore-loop each. The two pore-loops enter the membrane embedded part of the proteins from opposite sides of the membrane. The model was verified by accessibility studies of cysteine residues in single-Cys mutants of the Na+-glutamate transporter GltS of Escherichia coli, a member of the ESS family. Cysteine residues positioned in predicted periplasmic loops were accessible from the periplasm by a bulky, membrane-impermeable thiol reagent, while cysteine residues in cytoplasmic loops were not. Furthermore, two cysteine residues in the predicted pore-loop entering the membrane from the cytoplasmic side were shown to be accessible for small, membrane-impermeable thiol reagents from the periplasm, as was demonstrated before for the Na+-citrate transporter CitS of Klebsiella pneumoniae, a member of the 2HCT family. The data strongly suggests that GltS of the ESS family and CitS of the 2HCT family share the same fold as was predicted by comparing the averaged hydropathy profiles of the two families.     

Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop

Abstract

GltS of Escherichia coli is a secondary transporter that catalyzes Na⁺-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na⁺ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na⁺ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.     

Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis

To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.     

Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli

A genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described. The components of this system are the direct expression vector, pYZ4, and the mature beta-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein. The beta-subunit of sheep-kidney Na,K-ATPase (beta NKA), a class-II plasma membrane protein, was synthesized in E. coli using pYZ4, and BlaM was fused to a normally extracellular portion of it. The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane. A series of 31 beta NKA::BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane. This analysis revealed that the organisation of the beta NKA in the E. coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane.     

Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting.

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.     

The active conformation of glutamate synthase and its binding to ferredoxin

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.