Loop VIII/IX of the Na+-citrate transporter CitS of Klebsiella pneumoniae folds into an amphipathic surface helix

The sodium ion-dependent citrate transporter CitS of Klebsiella pneumoniae is a member of the 2-hydroxycarboxylate transporter (2HCT) family whose members transport divalent citrate in symport with two sodium ions. Profiles of the hydrophobic moment suggested the presence of an amphipathic helical structure in the cytoplasmic loop between transmembrane segments (TMSs) VIII and IX (the AH loop) in all members of the family. Cysteine-scanning mutagenesis was used to study the secondary structure of the AH loop. We have mutated 20 successive residues into cysteine residues, characterized each of the mutants for its transport activity, and determined the accessibility of the residues. Three of the mutants, G324C, F331C, and F332C, had very low citrate transport activity, and two others, I321C and S333C, exhibited significantly decreased activity after treatment of right-side-out membranes with membrane permeable thiol reagent N-ethylmaleimide (NEM), but not with membrane impermeable 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). No protection against NEM was observed with citrate or sodium ions. Labeling of the cysteine residues in the 20 mutants with the fluorescent probe fluorescein 5-maleimide, in membrane vesicles with an inverted orientation, resulted in a clear periodicity in the accessibility of the residues. Residues expected to be at the hydrophobic face of the putative alpha-helix were not accessible for the label, whereas those at the hydrophilic face were easily accessed and labeled. Pretreatment of whole cells and inside-out membranes expressing the mutants with the membrane impermeable reagent AmdiS confirmed the cytoplasmic localization of the AH region. It is concluded that the loop between TMSs VIII and IX folds into an amphipathic surface helix.     

Structure and elevator mechanism of the Na+-citrate transporter CitS

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Membrane topology of the Na(+)/citrate transporter CitS of Klebsiella pneumoniae by insertion mutagenesis

The sodium ion dependent citrate transporter of Klebsiella pneumoniae (CitS) is a member of the bacterial 2-hydroxycarboxylate transporter family. Membrane topology models of the protein, largely based on reporter molecule fusions to C-terminally truncated CitS molecules, indicate that the protein traverses the membrane 11 times with the NH(2)-terminus in the cytoplasm and the COOH-terminus in the periplasm. Furthermore, the structure is characterized by unusual long loops in the COOH-terminal half of the protein: one hydrophobic segment between transmembrane segments V and VI in the periplasm and three long loops connecting transmembrane segments VI and VII, VIII and IX and X and XI in the cytoplasm. The 10 kDa biotin acceptor domain and six consecutive His residues (His-tag) were inserted at different positions in the four long loops and the effect on transport activity and protein stability was analyzed. Six out of seven insertion mutants were stably expressed and three of these had retained significant transport activity. The sidedness of the tags in the mutants that tolerated the insertion was determined by proteolysis experiments. The results support the 11 transmembrane segment model that was based upon truncated CitS proteins.     

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.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.

The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three N-terminal and the six C-terminal membrane-spanning alpha-helices. However, the two additional helices that were predicted by hydropathy analyses between the N-terminal and C-terminal blocks did not conform with the fusion results. The analyses were therefore extended by probing the sideness of various engineered cysteine residues with the membrane-impermeant reagent 4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate. The results were in accord with those of the fusion analyses, suggesting that the protein folds within the membrane by a block of three N-terminal transmembrane segments and another one with six C-terminal transmembrane segments. The mainly hydrophobic connecting segment is predicted not to traverse the membrane fully, but to insert in an undefined manner from the periplasmic face. According to our model, the N-terminus is at the cytoplasmic face and the C-terminus is at the periplasmic face of the membrane.     

Specific modification of a Na+ binding site in NADH:quinone oxidoreductase from Klebsiella pneumoniae with dicyclohexylcarbodiimide

The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases Na+) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na+-translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N'-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for Na+ binding. At low Na+ concentrations (0.6 mM), DCCD inhibited both quinone reduction and Na+ transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM Na+, NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [14C]DCCD was prevented, indicating that Na+ and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue of the ND1 subunit from mitochondrial complex I. It is proposed that Na+ binds to the NuoH subunit during NADH-driven Na+ transport by NDH-1.     

The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters

The sodium solute symporters (SSS) and neurotransmitter sodium symporters (NSS) are two families of secondary transporters that are not related in amino acid sequence. Nonetheless, recent crystal structures showed that the Na⁺/galactose (SSS) and Na⁺/leucine (NSS) transporters have similar core structures. The structural relatedness highlights the need for classification methods for membrane protein structures based on other criteria than amino acid similarity. Here, we demonstrate that a method based on hydropathy profile alignments convincingly identifies structural similarity between the NSS and SSS families. Most importantly, the method shows that one of the largest transporter families for which a crystal structure is elusive (the amino acid/polyamine/organocation or APC superfamily), also shares the similar core structure observed for the Na⁺/galactose and Na⁺/leucine transporters. The APC superfamily contains the major amino acid transporter families that are found throughout life. Insight into their structure will significantly facilitate the studies of this important group of transporters.     

Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1

Removal of glutamate from the synaptic cleft by (Na(+) + K(+))-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30-100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.     

In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example

The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins provide rigid, hydrophilic surfaces for stable protein-protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins in vitro that are specific for the detergent-solubilized Na(+)-citrate symporter CitS of Klebsiella pneumoniae. Compared to classical hybridoma technology, the in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely.     

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.     

Solubilization and reconstitution of the Na(+)-dependent citrate carrier of Klebsiella pneumoniae

The citrate carrier of Klebsiella pneumoniae fermenting this substrate has been solubilized from the bacterial membranes with Triton X-100. The transport function was reconstituted by incorporation of the carrier into proteoliposomes using a freeze-thaw sonication procedure. Citrate uptake into these proteoliposomes required the presence of Na+ ions on the outside; the amount of citrate accumulated increased as the external Na+ concentration increased from 0 to 100 mM. Proteoliposomes preloaded with citrate catalyzed citrate counterflow when added to external [14C] citrate. Sodium ions were required for counterflow activity. The kinetics of citrate uptake, counterflow, or efflux were not influenced by an inside negative membrane potential, and the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone was without effect on citrate uptake. The data therefore suggest an electroneutral Na(+)-citrate symport mechanism for the transport of this tricarboxylic acid into K. pneumoniae.     

Characterization of the structure and function of Klebsiella pneumoniae allantoin racemase

The oxidative catabolism of uric acid produces 5-hydroxyisourate (HIU), which is further degraded to (S)-allantoin by two enzymes, HIU hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase. The intermediates of the latter two reactions, HIU and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, are unstable in solution and decay nonstereospecifically to allantoin. In addition, nonenzymatic racemization of allantoin has been shown to occur at physiological pH. Since the further breakdown of allantoin is catalyzed by allantoinase, an enzyme that is specific for (S)-allantoin, an allantoin racemase is necessary for complete and efficient catabolism of uric acid. In this work, we characterize the structure and activity of allantoin racemase from Klebsiella pneumoniae (KpHpxA). In addition to an unliganded structure solved using selenomethionyl single-wavelength anomalous dispersion, structures of C79S/C184S KpHpxA in complex with allantoin and with 5-acetylhydantoin are presented. These structures reveal several important features of the active site including an oxyanion hole and a polar binding pocket that interacts with the ureido tail of allantoin and serves to control the orientation of the hydantoin ring. The ability of KpHpxA to interconvert the (R)- and (S)-enantiomers of allantoin is demonstrated, and analysis of the steady-state kinetics of KpHpxA yielded a k_cat/K_m of 6.0 × 10⁵ M^− 1 s^− 1. Mutation of either of the active-site cysteines, Cys79 or Cys184, to serine inactivates this enzyme. The data presented provide new insights into the activity and substrate specificity of this enzyme and enable us to propose a mechanism for catalysis that is consistent with the two-base mechanism observed in other members of the aspartate/glutamate family.     

肺炎克雷伯菌blaCARB-2基因的分布及结构分析

为探讨β-内酰胺酶基因在临床分离肺炎克雷伯菌的分布及其相关可移动遗传元件的结构,本文利用基因组测序、PCR、分子克隆、接合转移和基因组学分析等方法,在对240株临床分离的肺炎克雷伯菌进行混合基因组测序的基础上,着重研究了bla_CARB-2等耐药性基因相关可移动遗传元件的结构及其在肺炎克雷伯菌基因组的定位,克隆获得耐药性质粒携带的若干耐药性基因并测定了它们的功能。结果显示,在240株肺炎克雷伯菌中检出11种β-内酰胺酶基因,其中1株肺炎克雷伯菌(KP1276)被检出bla_CARB-2基因,阳性率为0.42% (1/240);bla_CARB-2位于一个大小为182,450 bp的可接合转移质粒(pKP1276-182),pKP1276-182共编码222个基因,包含7个耐药性基因,分别是bla_CARB-2、bla_KLUC、aadA1、aadA2、cmlA1、dfrA1和sul2。bla_CARB-2是首次在肺炎克雷伯菌中被检出,且bla_CARB-2与其他3个耐药性基因一起构成一个新型结构的1型整合子(int-bla_CARB-2-aadA2-cmlA1-aadA1);对其中的3个耐药性基因bla_CARB-2、aadA2和CmlA1进行了分子克隆和耐药性测定,结果显示它们对相应药物都具有一定的耐药性, 其中bla_CARB-2对青霉素类β-内酰胺药物具有较高的耐药性。本研究结果表明,bla_CARB-2基因已经在肺炎克雷伯菌中出现,其由1型整合子携带编码位于一个可接合转移的质粒上,bla_CARB-2基因有可能在相同及不同种属肠杆菌之间进行水平转移,引起耐药性播散。     

Site-directed sulfhydryl labeling of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae: helix VIII comprises a portion of the sodium ion channel

Helix VIII of the beta-subunit of the oxaloacetate decarboxylase of Klebsiella pneumoniae contains the functionally important residues betaN373, betaG377, betaS382, and betaR389. Using a functional oxaloacetate decarboxylase mutant devoid of Cys residues in the beta-subunit, each amino acid residue in helix VIII was replaced individually with Cys. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 20 single-Cys replacement mutants with methanethiosulfonate (MTS) reagents in the absence or presence of Na(+) ions. The pattern of accessibility of the MTS reagents from the periplasmic side of helix VIII shows a periodicity which suggests that this region is alpha-helical. In particular, a water-accessible face comprising betaN373, betaG377, betaS382, betaM386, and betaV390 may be part of a Na(+) channel. Cys residues introduced in the cytoplasmically oriented part of helix VIII were accessible to three different water-soluble MTS compounds and therefore believed to be exposed to water on this side of the membrane. Most residues located in the upper part of helix VIII (residues betaN373-betaV381C) were protected by Na(+) ions for inactivation by the MTS reagents. The distinct results on accessibility toward the different MTS reagents obtained in the presence or absence of Na(+) ions may suggest a conformational change upon binding of Na(+) in this region. The betaR389C mutant had a reduced activity and a pH optimum at pH 9, which could be restored to a wild-type pH optimum of 6.5 and to a 400% gain in activity upon chemical modification with 2-aminoethyl methanethiosulfonate.     

Primary structure and characteristics of the melibiose carrier of Klebsiella pneumoniae.

The melB gene coding for the melibiose carrier of Klebsiella pneumoniae was cloned and sequenced. There were two potential translation initiation sites. It was predicted that the melibiose carrier consists of 471 (or 467) amino acid residues. Seventy-eight percent of the 471 amino acids were identical to the Escherichia coli melibiose carrier. Sugar transport characteristics were studied using an E. coli mel- mutant expressing cloned K. pneumoniae melB gene. Accumulation of melibiose via the K. pneumoniae melibiose carrier was not stimulated by adding NaCl or LiCl which stimulates melibiose accumulation via the E. coli melibiose carrier. Lactose was accumulated only in the presence of LiCl. TMG (methyl-1-thio-beta-D-galactopyranoside) was accumulated in the absence of added NaCl or LiCl. The accumulation was stimulated by LiCl but not by NaCl. Rapid H+ uptake was observed when melibiose or TMG was added to cell suspensions. These results suggest that the preferred cation couplings via K. pneumoniae melibiose carrier are H(+)-melibiose, Li(+)-lactose, and H+/Li(+)-TMG. This coupling spectrum is quite different from that of the E. coli melibiose carrier. It is of special interest that the K. pneumoniae melibiose carrier seems to be lacking the ability to recognize Na+ which is a preferred coupling cation of the E. coli melibiose carrier for all known sugar substrates. Further investigation of these two carriers may give us insight into the Na+ recognition site.     

NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae

Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO(3) (-2)) resistance is conferred by the ter (Te(r)) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven alpha-helices and a 3(10) helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 +/- 0.06 A for backbone atoms and 1.11 +/- 0.07 A for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique.