Cross-linking of dimeric CitS and GltS transport proteins

Abstract

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.     

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

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.     

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.     

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.     

Projection structure of the secondary citrate/sodium symporter CitS at 6 Å resolution by electron crystallography

CitS from Klebsiella pneumoniae acts as a secondary symporter of citrate and sodium ions across the inner membrane of the host. The protein is the best characterized member of the 2-hydroxycarboxylate transporter family, while no experimental structural information at sub-nanometer resolution is available on this class of membrane proteins. Here, we applied electron crystallography to two-dimensional crystals of CitS. Carbon-film-adsorbed tubular two-dimensional crystals were studied by cryo-electron microscopy, producing the 6-?-resolution projection structure of the membrane-embedded protein. In the p22(1)2(1)-symmetrized projection map, the predicted dimeric structure is clearly visible. Each monomeric unit can tentatively be interpreted as being composed of 11 transmembrane α-helices. In projection, CitS shows a high degree of structural similarity to NhaP1, the Na(+)/H(+) antiporter of Methanococcus jannaschii. We discuss possible locations for the dimer interface and models for the helical arrangements and domain organizations of the symporter based on existing models.     

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.     

Membrane insertion scanning of the human ileal sodium/bile acid co-transporter.

Mammalian sodium-dependent bile acid transporters (SBATs) responsible for bile salt uptake across the liver sinusoidal or ileal/renal brush border membrane have been identified and share approximately 35% amino acid sequence identity. Programs for prediction of topology and localization of transmembrane helices identify eight or nine hydrophobic regions for the SBAT sequences as membrane spanning. Analysis of N-linked glycosylation has provided evidence for an exoplasmic N-terminus and a cytoplasmic C-terminus, indicative of an odd number of transmembrane segments. To determine the membrane topography of the human ileal SBAT (HISBAT), an in vitro translation/translocation protocol was employed using three different fusion protein constructs. Individual HISBAT segments were analyzed for signal anchor or stop translocation (stop transfer) activity by insertion between a cytoplasmic anchor (HK M0) or a signal anchor segment (HK M1) and a glycosylation flag (HK beta). To examine consecutive HISBAT sequences, sequential hydrophobic sequences were inserted into the HK M0 vector or fusion vectors were made that included the glycosylated N-terminus of HISBAT, sequential hydrophobic sequences, and the glycosylation flag. Individual signal anchor (SA) and stop transfer (ST) properties were found for seven out of the nine predicted hydrophobic segments (H1, H2, H4, H5, H6, H7, and H9), supporting a seven transmembrane segment model. However, the H3 region was membrane inserted when translated in the context of the native HISBAT flanking sequences. Furthermore, results from translations of sequential constructs ending after H7 provided support for integration of H8. These data provide support for a SBAT transmembrane domain model with nine integrated segments with an exoplasmic N-terminus and a cytoplasmic C-terminus consistent with a recent predictive analysis of this transporter topology.     

Experimental evidence that the membrane-spanning helix of PufX adopts a bent conformation that facilitates dimerisation of the Rhodobacter sphaeroides RC-LH1 complex through N-terminal interactions

The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC-LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC-LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC-LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and β polypeptides on the other half of the dimer.     

Multimers of the precursor of a type IV pilin-like component of the general secretory pathway are unrelated to pili

Both the mature and precursor forms of PulG, a type IV pilin-like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross-linked into multimers similar to those obtained by cross-linking the components of type IV pili. To explore the possibility that the PulG precursor could form a pilus-like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of β-lactamase (βlaM) to the C-terminus. Neither of these changes affected multimerization. PulG precursor could be post-translationally processed by pre-pilin peptidase (PulO), indicating that the N-terminus of pre PulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme. Finally, precursor and mature forms of PulG could be efficiently cross-linked in a mixed dimer, indicating that at least a subpopu-lation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane. These results provide further evidence that the cross-linked multimers of the precursor form of PulG are unrelated to type IV pilus-like structures. It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus-like structure.     

Basic residues R260 and K357 affect the conformational dynamics of the major facilitator superfamily multidrug transporter LmrP

Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H(+)(Na(+)) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by components of the proton-motive force, the mutations also influence the energetics of transport.     

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.     

Purification and properties of glutamate synthase from Bacillus licheniformis

Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13](GltS) was purified to homogeneity from Bacillus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, approximately 158,000 and approximately 54,000). The enzyme was found to contain 8.1 +/- 1 iron atoms and 8.1 +/- 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GltS from B. licheniformis had a requirement for NADPH, alpha-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 microM. Nonhyperbolic kinetics were obtained for alpha-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 microM, and the apparent Km values obtained for alpha-ketoglutarate were 6 and 50 microM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DL-sulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding I0.5 values of 150, 11, and 250 microM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.     

Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy

The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.     

Co-assembly of envoplakin and periplakin into oligomers and Ca(2+)-dependent vesicle binding: implications for cornified cell envelope formation in stratified squamous epithelia

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca(2+). These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca(2+)] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.     

Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.     

The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. Roles for the KpsE and KpsD proteins

The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.     

Regions in the cytosolic C-terminus of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 are required for its homodimerization

The "secretory" Na+-K+-2Cl- cotransporter, NKCC1, is a member of a small gene family of electroneutral cation-chloride cotransporters (CCCs) with 9 homologues in vertebrates. A number of these transporters, including NKCC1 itself, have been shown to exist as homodimers in the membrane, suggesting that this may be a common feature of the CCCs. Here we employ chemical cross-linking studies, a novel co-immunoprecipition assay, and NKCC1/CCC chimeras to further explore the basis and significance of NKCC1 dimerization. An N-terminally truncated NKCC1 (nttNKCC1), in which the first 20 kDa of the 28 kDa cytosolic N-terminus are deleted, forms homodimers as well as heterodimers with full-length NKCC1, indicating that this region of N-terminus is not required for dimerization. On the other hand, replacing the 50 kDa NKCC1 C-terminus with that of several other non-NKCC1 homologues results in chimeric proteins that form homodimers but show little or no heterodimerization with NKCC1, demonstrating that the C-terminus of NKCC1 plays an essential role in dimerization and that NKCC1 dimerization exhibits definite homologue-specificity. Using additional chimeras we find that the residues required for dimer formation lie between amino acids 751 and 998 of (rat) NKCC1. We also show that dramatically overexpressing the nonfunctional truncated protein nttNKCC1 relative to the endogenous NKCC1 in the HEK293 cells results in a modest inhibition of fluxes via the endogenous transporter and a change in its sensitivity to the specific inhibitor bumetanide. These latter results indicate that there is a functional interaction between dimer subunits but that nonfunctional subunits do not necessarily have a dominant negative effect as has been previously proposed.     

Dimeric interaction between the cytoplasmic domains of the Na+/H+ exchanger NHE1 revealed by symmetrical intermolecular cross-linking and selective co-immunoprecipitation

To investigate the oligomeric structure of Na(+)/H(+) exchanger 1 (NHE1), permeabilized cells and membranes from cells expressing NHE1 variants were treated with the oxidizing agent Cu(2+)/o-phenanthroline or the bifunctional sulfhydryl reagent methanethiosulfonate. These treatments resulted in symmetrical intermolecular cross-linking at intrinsic (Cys(794) and Cys(561)) or 15 exogenous cysteine residues introduced into the distal carboxyl- (C-) terminal cytoplasmic domain (after aa 600) but not at intrinsic Cys(538) because of masking by its tight association with calcineurin B-homologous protein. Cross-linking was abolished in membranes solubilized with sodium dodecyl sulfate, which dissociates oligomeric NHE1, while it was preserved in those treated with Triton X-100. In addition, treatment with cross-linkers did not produce the tetrameric forms of NHE1 mutants with two cysteine residues. Thus, cross-linking presumably occurs between adjacent C-termini of the NHE1 dimer but not by a stochastic process via random collision of NHE1 molecules. The observations suggest that at least the distal C-termini of the NHE1 dimer are flexible or mobile and are thereby capable of easily making contact with each other over the large cytoplasmic portion of the molecule. Furthermore, co-immunoprecipitation experiments showed that the proximal C-termini (aa 503-580) have a strong propensity to interact directly with each other in parallel. Deletion of aa 562-579 resulted in disruption of disulfide cross-linking between the C-termini and markedly reduced the intracellular pH sensitivity of Na(+)/H(+) exchange, suggesting that the dimeric interaction in this region may control the pH-dependent regulation of NHE1.     

Solution structure of the transmembrane domain of the insulin receptor in detergent micelles

The insulin receptor (IR) binds insulin and plays important roles in glucose homeostasis by regulating the tyrosine kinase activity at its C-terminus. Its transmembrane domain (TMD) is shown to be important for transferring conformational changes induced by insulin across the cell membrane to regulate kinase activity. In this study, a construct IR_940–988 containing the TMD was expressed and purified for structural studies. Its solution structure in dodecylphosphocholine (DPC) micelles was determined. The sequence containing residues L962 to Y976 of the TMD of the IR in micelles adopts a well-defined helical structure with a kink formed by glycine and proline residues present at its N-terminus, which might be important for its function. Paramagnetic relaxation enhancement (PRE) and relaxation experimental results suggest that residues following the TMD are flexible and expose to aqueous solution. Although purified IR_940–988 in micelles existed mainly as a monomeric form verified by gel filtration and relaxation analysis, cross-linking study suggests that it may form a dimer or oligomers under micelle conditions.