Cysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helix

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys. Of the 26 single-Cys mutants, 16 accumulate xanthine to > or =50% of the steady state observed with C-less YgfO, 4 accumulate to low levels (10-25% of C-less), F322C, N325C, and N326C accumulate marginally (5-8% of C-less), and P318C, Q324C, and G340C are inactive. When transferred to wild type, F322C(wt) and N326C(wt) are highly active, but P318G(wt), Q324C(wt), N325C(wt), and G340C(wt) are inactive, and G340A(wt) displays low activity. Immunoblot analysis shows that replacements at Pro-318 or Gly-340 are associated with low or negligible expression in the membrane. More extensive mutagenesis reveals that Gln-324 is critical for high affinity uptake and ligand recognition, and Asn-325 is irreplaceable for active xanthine transport, whereas Thr-332 and Gly-333 are important determinants of ligand specificity. All single-Cys mutants react with N-ethylmaleimide, but regarding sensitivity to inactivation, they fall to three regions; positions 315-322 are insensitive to N-ethylmaleimide, with IC(50) values > or =0.4 mM, positions 323-329 are highly sensitive, with IC(50) values of 15-80 microM, and sensitivity of positions 330-340 follows a periodicity, with mutants sensitive to inactivation clustering on one face of an alpha-helix.     

Role of Intramembrane Polar Residues in the YgfO Xanthine Permease: HIS-31 AND ASN-93 ARE CRUCIAL FOR AFFINITY AND SPECIFICITY, AND ASP-304 AND GLU-272 ARE IRREPLACEABLE

Using the YgfO xanthine permease of Escherichia coli as a bacterial model for the study of the evolutionarily ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family, we performed a systematic Cys-scanning and site-directed mutagenesis of 14 putatively charged (Asp, Glu, His, Lys, or Arg) and 7 highly polar (Gln or Asn) residues that are predicted to lie in transmembrane helices (TMs). Of 21 single-Cys mutants engineered in the background of a functional YgfO devoid of Cys residues (C-less), only four are inactive or have marginal activity (H31C, N93C, E272C, D304C). The 4 residues are conserved throughout the family in TM1 (His-31), TM3 (Asn-93/Ser/Thr), TM8 (Glu-272), and putative TM9a (Asp-304/Asn/Glu). Extensive site-directed mutagenesis in wild-type background showed that H31N and H31Q have high activity and affinity for xanthine but H31Q recognizes novel purine bases and analogues, whereas H31C and H31L have impaired affinity for xanthine and analogues, and H31K or H31R impairs expression in the membrane. N93S and N93A are highly active but more promiscuous for recognition of analogues at the imidazole moiety of substrate, N93D has low activity, N93T has low affinity for xanthine or analogues, and N93Q or N93C is inactive. All mutants replacing Glu-272 or Asp-304, including E272D, E272Q, D304E, and D304N, are inactive, although expressed to high levels in the membrane. Finally, one of the 17 assayable single-Cys mutants, Q258C, was sensitive to inactivation by N-ethylmaleimide. The findings suggest that polar residues important for the function of YgfO cluster in TMs 1, 3, 8 and 9a.The nucleobase-ascorbate transporter (NAT)² or nucleobase-cation symporter-2 (NCS2) family is an evolutionarily ubiquitous family of purine, pyrimidine, and l-ascorbate transporters, with members specific for cellular uptake of uracil, xanthine, or uric acid (microbial and plant genomes) or vitamin C (mammalian genomes) (1, 2). Despite their importance for the recognition and uptake of several frontline purine-related drugs, NAT/NCS2 members have not been studied systematically at the molecular level, and high resolution structures or mechanistic models are missing. More than 1000 sequence entries are known, but few have been functionally characterized to date. The best studied eukaryotic member is UapA, a high affinity uric acid/xanthine:H⁺ symporter from the ascomycote Aspergillus nidulans (3–7). Studies with chimeric transporter constructs (3), site-directed mutagenesis, second-site suppressors, and kinetic inhibition analysis of ligand specificity have shown that a conserved NAT/NCS2 motif region between putative transmembrane helices 8 and 9 of UapA includes determinants of substrate recognition and selectivity, with at least one residue (Gln-408) implicated in binding with the imidazole moiety of purine (4), whereas a conserved QH motif at the middle of TM1 is important for activity and/or correct targeting to the plasma membrane (5), and an aromatic residue at the middle of TM12 (Phe-528) may act as a purine substrate selectivity filter (6). It has been proposed that TM1, TM12, and the NAT motif region interact functionally to determine affinity and specificity for uric acid (7).Recently, we characterized the first purine-specific members of the NAT/NCS2 family from a Gram-negative bacterium, namely YgfO and YicE of Escherichia coli K-12 (8), as high affinity xanthine:H⁺ symporters that cannot use uric acid, hypoxanthine, uracil, or other nucleobases as a substrate and cannot recognize analogues substituted at positions 7 or 8 of the imidazole ring. We launched a systematic series of Cys-scanning and site-directed mutagenesis studies of YgfO to elucidate structure-function relationships in a bacterial NAT (9, 10). In the course of these studies, we showed that the NAT motif sequence region of YgfO includes the essential determinants Gln-324, irreplaceable for high affinity binding and uptake; Asn-325, irreplaceable for active transport; and an α-helical stripe of residues (Thr-332, Gly-333, Ser-336, Val-339), highly sensitive to site-directed alkylation and important for ligand selectivity³ (9). In addition, we provided evidence that Asn-430 of TM12 is close to the purine binding site and Ile-432 optimizes binding indirectly (10). These studies also show that the bacterial (9, 10) and fungal (4, 6) NAT determinants are strikingly similar, implying that few of the residues conserved within the members of NAT family may be invariably critical for function.In this report, we have studied the highly polar (Gln or Asn) and putatively charged (Asp, Glu, His, Lys, or Arg) residues of YgfO permease that are predicted to lie in transmembrane helices. Such residues are expected to face other hydrophilic parts of the protein and/or the solvent-accessible environment of the binding pocket and often play crucial roles in substrate binding and the mechanism of energy coupling in active transport (11–14). Employing systematic site-directed mutagenesis of a set of 14 putatively charged and 7 highly polar residues predicted to lie in TMs (Fig. 1) and combining evidence from transport, immunoblotting, sulfhydryl alkylation, and ligand inhibition assays of a set of 60 site-directed mutants, we have identified four new important determinants in the YgfO mechanism: His-31 and Asn-93, which are crucial for affinity and/or specificity of binding purine analogues; and Glu-272 and Asp-304, which are irreplaceable for active xanthine transport. The results are discussed in conjunction with our previous findings on the role of TM12 and the NAT motif region and with respect to comparison with the major fungal homolog (UapA).Open in a separate windowFIGURE 1.Proposed topology of YgfO highlighting the polar/charged residues. This model is based on the program TMHMM, evidence that the C terminus is cytoplasmic (10, 25), and our unpublished evidence⁶ on the accessibility of loops to hydrophilic reagents from SCAM analysis. Putatively charged (K/R/H/D/E) or highly polar (Q/H) residues are enlarged and circled. Residues that fall in transmembrane helices (TMs) or in the NAT motif sequence (residues 323–333), as well as residue Asp-276 (which is discussed under “Discussion”) are shown with a dark background. Residues delineated as important to our studies are numbered and shown in red (this study) or blue (previous studies). The ambiguous topology segment 299–323 upstream of the NAT motif is designated as TM9a, and the transmembrane segment 330–357 that follows is designated as TM9b. SCAM analysis⁶ of the NAT motif shows that residues 323–333 are accessible to solvent from the outside (light blue-gray area), indicating that this region is topologically dynamic and might constitute a flexible, substrate-accessible (7, 9) reentry loop.     

Small, Acid-Soluble Spore Proteins of the α/β Type Do Not Protect the DNA in Bacillus subtilis Spores against Base Alkylation

Ethyl methanesulfonate (EMS) killed wild-type Bacillus subtilis spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the α/β type (α⁻β⁻ spores), and 20% of the survivors had obvious mutations. A recA mutation increased the EMS sensitivity of wild-type and α⁻β⁻ spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and α⁻β⁻ spore DNAs, in purified DNA, or in DNA saturated with α/β-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and α⁻β⁻ spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that α/β-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.     

Bacterial Cell Division Regulation: Lysogenization of Conditional Cell Division lon− Mutants of Escherichia coli by Bacteriophage Lambda

The lon⁻ mutants of Escherichia coli grow apparently normally except that, after temporary periods of inhibition of deoxyribonucleic acid synthesis, septum formation is specifically inhibited. Under these conditions, long, multinucleate, nonseptate filaments result. The lon⁻ mutation also creates a defect such that wild-type bacteriophage λ fails to lysogenize lon⁻ mutants efficiently and consequently forms clear plaques on a lon⁻ host. Two lines of evidence suggest that this failure probably results from interference with expression of the λcI gene, which codes for repressor, or with repressor action:-(i) when a lon⁻ mutant was infected with a λcII, cIII, or c Y mutant, there was an additive effect between the lon⁻ mutation and the λc mutations upon reduction of lysogenization frequency; and (ii) lon⁻ mutants permitted the growth of the λcro⁻ mutant under conditions in which the repressor was active. The isolation of λ mutants (λtp) which gained the ability to form turbid plaques on lon⁻ cells is also reported.     

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “Protaminobacter rubrum”

“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a ³²⁵RLDRD³²⁹ motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg³²⁵ and Arg³²⁸ in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R³²⁵Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter⁻¹ and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter⁻¹) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g⁻¹ with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.     

Cysteine-scanning Analysis of Helices TM8, TM9a,and TM9b and Intervening Loops in the YgfO Xanthine Permease: A CARBOXYL GROUP IS ESSENTIAL AT ASP-276

Bacterial and fungal members of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cys-scanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences ²⁵⁹FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV³¹⁴ and ³⁴²TIAVMLVILGLFP³⁵⁴ including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35–140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10–20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (K_i 79 μm) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC₅₀ 15 μm) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXGDXXAT) and in TM9a (GXXXDG).     

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys¹⁹-Pro²⁰-Trp²¹-Cys²² motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a “closed” compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an “open” conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.     

Targeted disruption of the Wnt regulator Kremen induces limb defects and high bone density

Kremen1 and Kremen2 (Krm1 and Krm2) are transmembrane coreceptors for Dickkopf1 (Dkk1), an antagonist of Wnt/β-catenin signaling. The physiological relevance of Kremen proteins in mammals as Wnt modulators is unresolved. We generated and characterized Krm mutant mice and found that double mutants show enhanced Wnt signaling accompanied by ectopic postaxial forelimb digits and expanded apical ectodermal ridges. Triple mutant Krm1^−/− Krm2^−/− Dkk1^+/− mice show enhanced growth of ectopic digits, indicating that Dkk1 and Krm genes genetically interact during limb development. Wnt/β-catenin signaling also plays a critical role in bone formation. Single Krm mutants show normal bone formation and bone mass, while double mutants show increased bone volume and bone formation parameters. Our study provides the first genetic evidence for a functional interaction of Kremen proteins with Dkk1 as negative regulators of Wnt/β-catenin signaling and reveals that Kremen proteins are not universally required for Dkk1 function.     

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml⁻¹, 2 μg of erythromycin ml⁻¹, 30 μg of chloramphenicol ml⁻¹, or 1 μg of tetracycline ml⁻¹ or a combination of 300 μg of streptomycin ml⁻¹ and 150 μg of spectinomycin ml⁻¹. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10⁻⁷ were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10⁻³ were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

Stacking Interactions between Carbohydrate and Protein Quantified by Combination of Theoretical and Experimental Methods

Carbohydrate – receptor interactions are an integral part of biological events. They play an important role in many cellular processes, such as cell-cell adhesion, cell differentiation and in-cell signaling. Carbohydrates can interact with a receptor by using several types of intermolecular interactions. One of the most important is the interaction of a carbohydrate''s apolar part with aromatic amino acid residues, known as dispersion interaction or CH/π interaction. In the study presented here, we attempted for the first time to quantify how the CH/π interaction contributes to a more general carbohydrate - protein interaction. We used a combined experimental approach, creating single and double point mutants with high level computational methods, and applied both to Ralstonia solanacearum (RSL) lectin complexes with α-l-Me-fucoside. Experimentally measured binding affinities were compared with computed carbohydrate-aromatic amino acid residue interaction energies. Experimental binding affinities for the RSL wild type, phenylalanine and alanine mutants were −8.5, −7.1 and −4.1 kcal.mol⁻¹, respectively. These affinities agree with the computed dispersion interaction energy between carbohydrate and aromatic amino acid residues for RSL wild type and phenylalanine, with values −8.8, −7.9 kcal.mol⁻¹, excluding the alanine mutant where the interaction energy was −0.9 kcal.mol⁻¹. Molecular dynamics simulations show that discrepancy can be caused by creation of a new hydrogen bond between the α-l-Me-fucoside and RSL. Observed results suggest that in this and similar cases the carbohydrate-receptor interaction can be driven mainly by a dispersion interaction.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β₂ integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC³³⁶ (³³³LEEYSKR³³⁹) is highly conserved among RTX toxins, whereas CRAC⁵⁰³ (⁵⁰¹VDYLK⁵⁰⁵) is unique to LtxA. A peptide corresponding to CRAC³³⁶ inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC³³⁶ and CRAC⁵⁰³ bind cholesterol, only CRAC³³⁶ competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC³³⁶ site was essential for LtxA cytotoxicity. The conservation of CRAC³³⁶ among RTX toxins suggests that this mechanism may be conserved among RTX toxins.     

A New Class of Endoplasmic Reticulum Export Signal ΦXΦXΦ for Transmembrane Proteins and Its Selective Interaction with Sec24C

Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), ²⁶VSIPM³⁰ in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues ⁸⁹⁵LIL⁸⁹⁷ (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to ⁸⁹⁵AAA⁸⁹⁷, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.     

Novel Reporter Alleles of GSK-3α and GSK-3β

Glycogen Synthase Kinase 3 (GSK-3) is a key player in development, physiology and disease. Because of this, GSK-3 inhibitors are increasingly being explored for a variety of applications. In addition most analyses focus on GSK-3β and overlook the closely related protein GSK-3α. Here, we describe novel GSK-3α and GSK-3β mouse alleles that allow us to visualise expression of their respective mRNAs by tracking β-galactosidase activity. We used these new lacZ alleles to compare expression in the palate and cranial sutures and found that there was indeed differential expression. Furthermore, both are loss of function alleles and can be used to generate homozygous mutant mice; in addition, excision of the lacZ cassette from GSK-3α creates a Cre-dependent tissue-specific knockout. As expected, GSK3α mutants were viable, while GSK3β mutants died after birth with a complete cleft palate. We also assessed the GSK-3α mutants for cranial and sternal phenotypes and found that they were essentially normal. Finally, we observed gestational lethality in compound GSK-3β^−/−; GSK3α^+/− mutants, suggesting that GSK-3 dosage is critical during embryonic development.     

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (K_d = 34 μm) to aquo-cobalamin (H₂OCbl⁺) but not to other cobalamins. Competition experiments with the H₂OCbl⁺-coordinating ligand CN⁻ and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H₂OCbl⁺ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H₂OCbl⁺. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins.