Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

The structure of the membrane integral rotor ring of the proton translocating F₁F₀ ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c₁₁ rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6–10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu⁶¹ in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu⁶¹ is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu⁶¹ by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.ATP synthases found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts catalyze ATP synthesis and ATP hydrolysis coupled with transmembrane proton or sodium ion transport. The enzymes are multi-subunit complexes composed of an extra-membranous catalytic F₁ domain and an interconnected integral membrane F₀ domain. The hydrophilic F₁ domain consists of five different polypeptides with a stoichiometry of α₃β₃γδϵ. Detailed structural information obtained with the mitochondrial enzyme (1–3) in combination with biochemical (4), biophysical (5), and single molecule studies (6–9) revealed that synthesis or hydrolysis of ATP in the F₁ domain is accomplished via a rotary catalytic mechanism. In addition to information on the catalytic mechanism, structure analysis and single molecule studies of the mitochondrial or the chloroplast F₁ complex have also unraveled the molecular mechanism of several F₁-specific inhibitors (10–14). Less detailed information is available on the integral membrane F₀ domain, which consists of three different polypeptides (a, b, and c) and mediates the transfer of protons or sodium ions across the membrane. Subunits a and b were shown to reside at the periphery of a cylindrical complex formed by multiple copies of the c subunit (15–18). The number of c subunits in the cylindrical subcomplex shows substantial variation in different organisms. Ten protomers are found in ATP synthases from yeast, Escherichia coli and Bacillus PS3 (19–21), 11 in Ilyobacter tartaricus, Propionigenium modestum, and Clostridium paradoxum (22–24), 13 in the thermoalkalophilic Bacillus TA2.TA1 (25), 14 in spinach chloroplasts (26), and 15 in the cyanobacterium Spirulina platensis (27). The structure of isolated subunits a, b, and c from E. coli has been studied by mutagenesis analysis and by NMR spectroscopy in a mixed solvent that was suggested to mimic the membrane environment (28–32). These studies showed that subunit a folds with five membrane-spanning helices. The fourth of these helices directly interacts with subunit c and contains a conserved arginine (Arg²¹⁰), which is thought to be involved in proton transfer (33). Subunit b, which is present in two copies in the intact F₀, contains a single transmembrane helix. Cross-linking data support a direct interaction of the two copies of the b subunit (29). Subunit c was studied at two different pH values to obtain the protonated and deprotonated form of a conserved carboxylate (Asp⁶¹ in E. coli) that was shown to be essential for proton transport (34). NMR spectroscopy revealed that the isolated c subunit consists of two long hydrophobic membrane spanning segments connected by a short hydrophilic loop (30, 35). This loop is located close to the γ and ϵ subunit on the F₁ side of the membrane (36, 37). Low resolution x-ray crystallography, cryo-electron microscopy, and atomic force microscopy showed that the membrane-spanning helices of the multiple copies of subunit c in the intact F₀ complex are tightly packed in two concentric rings (19, 22, 26). Atomic resolution of the c ring was recently provided for the Na⁺-translocating F-type ATPase from I. tartaricus (38) and the related Na⁺-translocating V-type ATPase from Enterococcus hirae (39). Rotation of the c ring was demonstrated by cross-linking (18), fluorescence studies (40), and single molecule visualization (41, 42). Based on the structural and biochemical information on F₁ and F₀, different mechanical models have been proposed describing how the rotation of the c ring is coupled to the rotation of the F₁ rotor subunits. This rotation in turn drives sequential conformational shifts at the three catalytic β subunits that result in ATP synthesis (43–45). Vice versa hydrolysis of ATP in the F₁ domain is thought to drive rotation of the γϵc_10–15 subcomplex and transports protons or sodium ions across the membrane.Here we describe the crystal structure of the chloroplast c₁₄ rotor, which is the first structure of an isolated c ring rotor from a proton driven ATPase. The structure was solved by molecular replacement using a tetradecameric search model that was generated from a monomer taken from the I. tartaricus c₁₁ structure. The imposition of noncrystallographic symmetry restraints during refinement substantially improved electron density and structure determination.     

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca²⁺-activated Cl⁻ channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y₂ receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca²⁺ and a Ca²⁺-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl⁻ conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I_Cl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl⁻ channels and may also have a function during proliferation and apoptotic cell death.Regulation of cell volume is fundamental to all cells, particularly during cell growth and division. External hypotonicity leads to cell swelling and subsequent activation of volume-regulated chloride and potassium channels, to release intracellular ions and to re-shrink the cells, a process termed regulatory volume decrease (RVD)³ (1). Volume-regulated chloride currents (I_Cl,swell) have dual functions during cell proliferation as well as apoptotic volume decrease (AVD), preceding apoptotic cell death (2). Although I_Cl,swell is activated in swollen cells to induce RVD, AVD takes place under normotonic conditions to shrink cells (3, 4). Early work suggested intracellular Ca²⁺ as an important mediator for activation of I_Cl,swell and volume-regulated K⁺ channels (5), whereas subsequent studies only found a permissive role of Ca²⁺ for activation of I_Cl,swell (6), reviewed in Ref. 1. In addition, a plethora of factors and signaling pathways have been implicated in activation of I_Cl,swell, making cell volume regulation an extremely complex process (reviewed in Refs. 1, 3, and 7). These factors include intracellular ATP, the cytoskeleton, phospholipase A2-dependent pathways, and protein kinases such as extracellular-regulated kinase ERK1/2 (reviewed in Refs. 1 and 7). Previous approaches in identifying swelling-activated Cl⁻ channels have been unsuccessful or have produced controversial data. Thus none of the previous candidates such as pI_Cln, the multidrug resistance protein, or ClC-3 are generally accepted to operate as volume-regulated Cl⁻ channels (reviewed in Refs. 8 and 9). Notably, the cystic fibrosis transmembrane conductance regulator (CFTR) had been shown in earlier studies to influence I_Cl,swell and volume regulation (10–12). The variable properties of I_Cl,swell suggest that several gene products may affect I_Cl,swell in different cell types.The TMEM16 transmembrane protein family consists of 10 different proteins with numerous splice variants that contain 8–9 transmembrane domains and have predicted intracellular N- and C-terminal tails (13, 16–18). TMEM16A (also called ANO1) is required for normal development of the murine trachea (14) and is associated with different types of tumors, dysplasia, and nonsyndromic hearing impairment (13, 15). TMEM16A has been identified as a subunit of Ca²⁺-activated Cl⁻ channels that are expressed in epithelial and non-epithelial tissues (16–18). Interestingly, members of the TMEM16 family have been suggested to play a role in osmotolerance in Saccharomyces cerevisiae (19). Here we show that TMEM16 proteins also contribute to I_Cl,swell and regulatory volume decrease.     

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

The reduction of nitrite (NO₂⁻) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H₂O and D₂O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H₂O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mm. As a consequence, the observed ET process was faster in D₂O than in H₂O above 0.1 mm nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10 mm. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.Denitrification is an anaerobic respiration pathway found in bacteria, archaea, and fungi, in which ATP synthesis is coupled to the sequential reduction of nitrate (NO₃⁻) and nitrite (NO₂⁻) (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (1–3).³ The first committed step in this reaction cascade is the formation of gaseous NO by nitrite reductase (NiR), the key enzyme of this pathway. Two distinct classes of periplasmic NiR are found in denitrifying bacteria, one containing cd₁ hemes as prosthetic groups (4–6) and the other utilizing two copper centers to catalyze the one-electron reduction of nitrite (7). Copper-containing NiRs are divided into two main groups according to the color of their oxidized type 1 copper center (T1Cu), with shades ranging from blue to green (3, 7). NiR from Alcaligenes xylosoxidans subsp. xylosoxidans (NCIMB 11015, AxNiR), which is analyzed in this study, is a member of the blue CuNiR group. The blue and green subclasses show a high degree of sequence similarity (70%) (8) and have similar trimeric structures with each monomer (∼36.5 kDa in AxNiR) consisting of two greek key β-barrel cupredoxin-like motifs as well as one long and two short α-helical regions (7, 9).Each NiR monomer contains two copper-binding sites per catalytic unit. One is a T1Cu center, which receives electrons from a physiological redox partner protein and is buried 7 Å beneath the protein surface (10), and the other copper is a type 2 center (T2Cu), constituting the catalytically active substrate-binding site (11). The physiological electron donor for the blue NiRs are the small copper protein azurin (14 kDa) (7) and cytochrome c₅₅₁ (7, 12, 13). The T1Cu, which is responsible for the color of NiR, serves as the electron delivery center and is coordinated by two histidine residues as well as one cysteine and one methionine residue. The catalytic T2Cu, which like all T2Cu centers has very weak optical bands, is ligated to three His residues and an H₂O/OH⁻ ligand in the resting state. This H₂O/OH⁻ ligand is held in place by hydrogen bonds to the active site residues, Asp-92 (AxNiR numbering) and His-249, and gets displaced by the substrate during catalytic turnover (14). The T2Cu is located at the base of a 13–14-Å substrate access channel at the interface of two monomers with one of the three His residues being part of the adjacent subunit (15, 16). The two copper centers are connected by a 12.6-Å covalent bridge provided by the T1Cu-coordinating Cys and by one of the T2Cu His ligands (17, 18). This linkage has been suggested to constitute the electron transfer (ET) pathway from the T1Cu center to the catalytically active T2Cu center via 11 covalent bonds (19).Intramolecular ET from T1- to T2Cu has been extensively examined using pulse radiolysis studies (7, 19–24). In a variety of NiR species, ET could be measured, both in the presence and absence of substrate, with observed ET rate constants (k_ET(obs)) ranging from ∼150 to ∼2000 s⁻¹. According to the Marcus semi-classical ET theory (25), the redox potentials (E′₀, redox midpoint potential at pH 7.0) of the copper centers affect both the thermodynamic equilibrium and the ET kinetics. In the absence of substrate, the difference in the redox potentials has been found to be insignificant at pH 7 (E′₀ (T1Cu) ∼240 mV and E′₀ (T2Cu) ∼230 mV (20)), implying a thermodynamically equal electron distribution between the two metal centers. From an enzymatic point of view, however, approaching this equilibrium position on such a fast time scale (≥150 s⁻¹) is unfavorable in the absence of substrate, as NiR has been shown to form an inactive species with a reduced T2Cu that is devoid of the H₂O/OH⁻ ligand and unable to bind nitrite (26, 27). Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7, 16, 25, 27–30). However, k_ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitrite-bound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).In addition to changes in the redox potentials and thus in the driving force of the ET reaction, several structural changes in the redox centers have been reported as a result of substrate binding, which may also influence the inter-copper ET rate by changing the reorganization energy (16, 25, 30, 31). These rearrangements include subtle changes in the Cys-His bridge linking T1- and T2Cu (32) and conformational transitions of the catalytically relevant active site residue Asp-92 (see below and Ref. 29). Moreover, the presence of nitrite has been postulated to be relayed to the T1Cu site via the so-called substrate sensor loop (via His-94, Asp-92, and His-89 in AxNiR), thereby triggering ET to the T2Cu (19, 27, 29, 32). The tight coupling of ET to the presence of substrate has been argued to prevent the formation of a deactivated enzyme species with a prematurely reduced T2Cu (14, 16, 19, 26, 27, 33). In accordance with such a feedback mechanism, in a combined crystallographic and single-crystal spectroscopic study, inter-copper ET could only be detected in crystals where nitrite was bound to the T2Cu site, whereas in the absence of substrate no such ET was observed (34). This finding, however, contradicts the pulse radiolysis results at room temperature (see above), and the apparent discrepancy between solution studies and x-ray crystallographic data collected at cryogenic temperature remains to be resolved.The one-electron reduction of nitrite to NO involves two protons according to the chemical net equation NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O, if the T2Cu is ligated by an H₂O molecule in the resting state rather than an OH⁻ ion. Although the exact enzymatic mechanism is still somewhat controversial (35, 36), one suggested reaction sequence is given in Scheme 1. The potential participation of active site residues in catalyzing the proton transfer (PT) steps has been investigated by studying the pH dependence of NiR under steady-state conditions as well as by pulse radiolysis. The trends obtained for k_cat and k_ET(obs), are similar with pH optima between 5.2 and 6, indicating the involvement of two amino acid residues (21, 22, 37). Asp-92 and His-249 have been proposed as acid-base catalysts (18, 21, 22, 28, 38), and the abrupt drop in rates at increasing pH may indicate that OH⁻ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35, 40, 41).⁴Open in a separate windowSCHEME 1.A potential reaction mechanism proposed for CuNiRs. Adapted from Ref. 36. Nitrite is shown to bind to the oxidized T2Cu as nitrous acid, thus involving the first protonation step. It coordinates to the oxidized T2Cu center in a bidentate fashion. Following inter-copper ET yielding a reduced T2Cu center, the initially deprotonated Asp-92 accepts a proton, which is subsequently transferred to the substrate. His-249 may be a potential source of this second proton. PT and ET reactions may be reversible and they may be concerted rather than sequential as suggested by the arrows. See text for further information.There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR. In this study of the blue AxNiR, our aims were to gain further insight into the mechanism of nitrite reduction by combining multiple turnover experiments with laser photoexcitation studies to measure the (single turnover) inter-copper ET. An extensive analysis of the solvent kinetic isotope effect (SKIE) has been employed as a means of determining whether solvent-exchangeable protons and/or water molecules play a rate-limiting role in the catalytic turnover and/or in inter-copper ET.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

Protein-tyrosine Phosphatase-�� and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling

Calcium (Ca²⁺) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca²⁺ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca²⁺ release channel IP₃R. In response to IL-1, catalytically active PTPα was required for Ca²⁺ release from the ER, Src-dependent phosphorylation of IP₃R1 and accumulation of IP₃R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP₃R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca²⁺ signaling.The interleukin-1 (IL-1)³ family of pro-inflammatory cytokines mediates host responses to infection and injury. Impaired control of IL-1 signaling leads to chronic inflammation and destruction of extracellular matrices (1, 2), as seen in pathological conditions such as pulmonary fibrosis (3), rheumatoid arthritis (4, 5), and periodontitis (6). IL-1 elicits multiple signaling programs, some of which trigger Ca²⁺ release from the endoplasmic reticulum (ER) as well as expression of multiple cytokines and inflammatory factors including c-Fos and c-Jun (7, 8), and matrix metalloproteinases (9, 10), which mediate extracellular matrix degradation via mitogen-activated protein kinase-regulated pathways (11).In anchorage-dependent cells including fibroblasts and chondrocytes, focal adhesions (FAs) are required for IL-1-induced Ca²⁺ release from the ER and activation of ERK (12–14). FAs are actin-enriched adhesive domains composed of numerous (>50) scaffolding and signaling proteins (15–17). Many FA proteins are tyrosine-phosphorylated, including paxillin, focal adhesion kinase, and src family kinases, all of which are crucial for the assembly and disassembly of FAs (18–21). Protein-tyrosine phosphorylation plays a central role in regulating many cellular processes including adhesion (22, 23), motility (24), survival (25), and signal transduction (26–29). Phosphorylation of proteins by kinases is balanced by protein-tyrosine phosphatases (PTP), which can enhance or attenuate downstream signaling by dephosphorylation of tyrosine residues (30–32).PTPs can be divided into two main categories: receptor-like and intracellular PTPs (33). Two receptor-like PTPs have been localized to FA (leukocyte common antigen-related molecule and PTPα). Leukocyte common antigen-related molecule can dephosphorylate and mediate degradation of p130^cas, which ultimately leads to cell death (34, 35). PTPα contains a heavily glycosylated extracellular domain, a transmembrane domain, and two intracellular phosphatase domains (33, 36). The amino-terminal domain predominantly mediates catalytic activity, whereas the carboxyl-terminal domain serves a regulatory function (37, 38). PTPα is enriched in FA (23) and is instrumental in regulating FA dynamics (39) via activation of c-Src/Fyn kinases by dephosphorylating the inhibitory carboxyl tyrosine residue, namely Tyr⁵²⁹ (22, 40–42) and facilitation of integrin-dependent assembly of Src-FAK and Fyn-FAK complexes that regulate cell motility (43). Although PTPα has been implicated in formation and remodeling of FAs (44, 45), the role of PTPα in FA-dependent signaling is not defined.Ca²⁺ release from the ER is a critical step in integrin-dependent IL-1 signal transduction and is required for downstream activation of ERK (13, 46). The release of Ca²⁺ from the ER depends on the inositol 1,4,5-triphosphate receptor (IP₃R), which is an IP₃-gated Ca²⁺ channel (47). All of the IP₃R subtypes (subtypes 1–3) have been localized to the ER, as well as other the plasma membrane and other endomembranes (48–50). Further, IP₃R may associate with FAs, enabling the anchorage of the ER to FAs (51, 52). However, the molecule(s) that provide the structural link for this association has not been defined.FA-restricted, IL-1-triggered signal transduction in anchorage-dependent cells may rely on interacting proteins that are enriched in FAs and the ER (53). Here, we examined the possibility that PTPα associates with c-Src and IP₃R to functionally link FAs to the ER, thereby enabling IL-1 signal transduction.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors

The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic “proton switch.” Infrared and fluorescence spectroscopic pK_a determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pK_a into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors.G protein-coupled receptors (GPCRs)² are hepta-helical membrane proteins that couple a large variety of extracellular signals to cell-specific responses via activation of G proteins. In the visual photoreceptor rhodopsin, a prototypical class I GPCR (1, 2), molecular activation processes can be monitored in real time by spectroscopic assays and analyzed in the context of several crystal structures (3–8). The primary signal for rhodopsin is the 11-cis to all-trans photoisomerization of retinal covalently bound to the apoprotein opsin through a protonated Schiff base to Lys²⁹⁶. Current models converge toward a picture in which “microdomains” act as conformational switches that are coupled to different degrees to the primary activation process. Two activating “proton switches” have been identified (9) as follows: breakage of an intramolecular salt bridge (10) by transfer of the Schiff base proton to its counter ion Glu-113 (11) is followed by movement of helix-6 (H6) (12, 13) in the metarhodopsin II_a (MII_a) to MII_b transition. The MII_b state takes up a proton at Glu-134 (14) in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MII_bH⁺ intermediate (15, 16), which activates transducin (G_t), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17) in membranes as reviewed previously (18), and in complex with G_t the protonated state of the carboxyl group becomes stabilized (19). This charge alteration is linked to the release of an “ionic lock,” originally described for the β₂-adrenergic receptor (20), which also in rhodopsin stabilizes the inactive state (16) through interactions between the cytosolic ends of H3 and H6 (21).In the absence of a lipidic bilayer, proton uptake and H6 movement become uncoupled (15). Lipidic composition affects MII formation, rhodopsin structure, and oligomerization (22–24) and differs at the rhodopsin membrane interface from the bulk lipidic phase (25). Likewise, MII formation specifically affects lipid structure (26). Although of fundamental importance for GPCR activation, the potential implication of lipid protein interactions in “proton switching” is not clear. A functional role of Glu-134 in lipid interactions has been originally derived from IR spectra where E134Q replacement abolished changes of lipid headgroup vibrations in the MIIG_t complex (19). Computational approaches emphasized the “strategic” location of the D(E)RY motif (27), and the Glu-134 carboxyl pK_a may critically depend on the lipid protein interface (28). However, the implications for proton switching are not evident, and the theoretical interest is contrasted by the lack of experimental data addressing the effect of the lipidic phase on side chain protonation, secondary structure, and membrane topology of the D(E)RY motif.We have studied the coupling between conformation and protonation in single transmembrane segments derived from H3 of bovine rhodopsin. We have assessed the “modular” function of the D(E)RY motif by determining parameters not evident from the crystal structures, i.e. the pK_a of the conserved carboxyl, its linkage to helical structure, and the effect of protonation on side chain to lipid headgroup distance. We show that the D(E)RY motif encodes an autonomous “proton switch” controlling side chain exposure and helix formation in the low dielectric of a lipidic phase. The data ascribe a functional role to lipid protein interactions that couple the chemical potential of protons to an activity-promoting GPCR conformation in a ligand-independent manner.     

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN^{K252 + 255A}, SEN^K304A, SEN^K334A, SEN^K344E, SEN^K435L, and SEN^Δ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN^K344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN^K344E had lost α-enolase activity, which was also reduced in SEN^K334A and SEN^Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN^{K252 + 255A}, SEN^K435L, and SEN^Δ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.Streptococcus pyogenes (group A Streptococcus, GAS)⁸ is a common bacterial pathogen, causing over 700 million human disease episodes each year (1). These range from serious life-threatening invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome to non-invasive infections like pharyngitis and pyoderma. Invasive disease, in combination with postinfection immune sequelae including rheumatic heart disease and acute poststreptococcal glomerulonephritis, account for over half a million deaths each year (1). Although a resurgence of GAS invasive infections has occurred in western countries since the mid-1980s, disease burden is much greater in developing countries and indigenous populations of developed nations, where GAS infections are endemic (2–4).GAS is able to bind human plasminogen and activate the captured zymogen to the serine protease plasmin (5–17). The capacity of GAS to do this plays a critical role in virulence and invasive disease initiation (3, 17–19). The plasminogen activation system in humans is an important and highly regulated process that is responsible for breakdown of extracellular matrix components, dissolution of blood clots, and cell migration (20, 21). Plasminogen is a 92-kDa zymogen that circulates in human plasma at a concentration of 2 μm (22). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg⁵⁶¹–Val⁵⁶² site (23), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (20, 23). GAS also has the ability to activate human plasminogen by secreting the virulence determinant streptokinase. Streptokinase forms stable complexes with plasminogen or plasmin, both of which exhibit plasmin activity (20, 24). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α₂-antiplasmin and α₂-macroglobulin (11, 20). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (6). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (25), the PAM-related protein (19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (9, 26), and streptococcal surface enolase (SEN or α-enolase) (27). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (6).In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (27–29). SEN is abundantly expressed in the cytosol of most bacterial species but has also been identified as a surface-located protein in GAS and other bacteria including pneumococci, despite lacking classical cell surface protein motifs such as a signal sequence, membrane-spanning domain, or cell-wall anchor motif (27, 28, 30, 31). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (27, 32). In contrast, an internal binding motif containing lysines at positions 252 and 255 in the closely related α-enolase of Streptococcus pneumoniae has been shown to play a pivotal role in the acquisition of plasminogen in this bacterial species (33). The octameric pneumococcal α-enolase structure consists of a tetramer of dimers. Hence, potential binding sites could be buried in the interface between subunits. In fact, the crystal structure of S. pneumoniae α-enolase revealed that the two C-terminal lysine residues are significantly less exposed than the internal plasminogen-binding motif (34).In this study, we constructed an in silico model of GAS SEN, based on the pneumococcal octameric α-enolase crystal structure, and validated this model using ion mobility (IM) mass spectrometry (MS). Site-directed mutagenesis followed by structural and functional analyses revealed that Lys³⁴⁴ plays a crucial role in structural integrity and enzymatic function. Furthermore, we demonstrate that the plasminogen-binding motif residues Lys²⁵² and Lys²⁵⁵ and the C-terminal Lys⁴³⁴ and Lys⁴³⁵ residues are located adjacently in the GAS SEN structure and play a concerted role in the binding of human plasminogen.     

Adipose Triglyceride Lipase Is Implicated in Fuel- and Non-fuel-stimulated Insulin Secretion

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous β-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL^−/− mice indicated the presence of other TG lipase(s) in the β-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K_ATP-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL^−/− mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL^−/− mice. Accordingly, isolated islets from ATGL^−/− mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL^−/− islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.Free fatty acids (FFA)⁵ and other lipid molecules are important for proper glucose-stimulated insulin secretion (GSIS) by β-cells. Thus, deprivation of fatty acids (FA) in vivo (1) diminishes GSIS, whereas a short term exposure to FFA enhances it (1–3). In contrast, a sustained provision of FA, particularly in the presence of high glucose in vitro, is detrimental to β-cells in that it reduces insulin gene expression (4) and secretion (5) and induces β-cell apoptosis (6). The FA supply to the β-cells can be from exogenous sources, such as plasma FFAs and lipoproteins, or endogenous sources, such as intracellular triglyceride (TG) stores. Studies from our laboratory (7–10) and others (11, 12) support the concept that the hydrolysis of endogenous TG plays an important role in fuel-induced insulin secretion because TG depletion with leptin (13) or inhibition of TG lipolysis by lipase inhibitors such as 3,5-dimethylpyrazole (7) or orlistat (11, 12) markedly curtail GSIS in rat islets. Furthermore, mice with β-cell-specific knock-out of hormone-sensitive lipase (HSL), which hydrolyzes both TG and diacylglycerol (DAG), show defective first phase GSIS in vivo and in vitro (14).Lipolysis is an integral part of an essential metabolic pathway, the TG/FFA cycle, in which FFA esterification onto a glycerol backbone leading to the synthesis of TG is followed by its hydrolysis with the release of the FFA that can then be re-esterified. Intracellular TG/FFA cycling is known to occur in adipose tissue of rats and humans (15, 16) and also in liver and skeletal muscle (17). It is generally described as a “futile cycle” as it leads to the net hydrolysis of ATP with the generation of heat (18). However, several studies have shown that this cycle has important functions in the cell. For instance, in brown adipose tissue, it contributes to overall thermogenesis (17, 19). In islets from the normoglycemic, hyperinsulinemic, obese Zucker fatty rat, increased GSIS is associated with increased glucose-stimulated lipolysis and FA esterification, indicating enhanced TG/FFA cycling (10). Stimulation of lipolysis by glucose has also been observed in isolated islets from normal rats (12) and HSL^−/− mice (8) indicating the presence of glucose-responsive TG/FFA cycling in pancreatic β-cells.The identity of the key lipases involved in the TG/FFA cycle in pancreatic islets is uncertain. HSL is expressed in islets (20), is up-regulated by long term treatment with elevated glucose (21), and is associated with insulin secretory granules (22). In addition, our earlier results suggested that elevated HSL expression correlates with augmented TG/FFA cycling in islets of Zucker fatty rats (10). However, it appears that other lipases may contribute to lipolysis and the regulation of GSIS in islet tissue. Thus, results from studies using HSL^−/− mice showed unaltered GSIS (8, 23), except in fasted male mice (8, 9) in which lipolysis was decreased but not abolished. Furthermore, HSL^−/− mice show residual TG lipase activity (8) indicating the presence of other TG lipases.Recently, adipocyte triglyceride lipase (ATGL; also known as Desnutrin, TTS-2, iPLA₂-ζ, and PNPLA2) (24–26) was found to account for most if not all of the residual lipolysis in HSL^−/− mice (26, 27). Two homologues of ATGL, Adiponutrin and GS2, have been described in adipocytes (24). All three enzymes contain a patatin-like domain with broad lipid acyl-hydrolase activity. However, it is not known if adiponutrin and GS2 are actually TG hydrolases. An additional lipase, TG hydrolase or carboxylesterase-3, has been identified in rat adipose tissue (28, 29). Although the hydrolysis of TG is catalyzed by all these lipases, HSL can hydrolyze both TG and DAG, the latter being a better substrate (30).In this study, we observed that besides HSL, ATGL (31), adiponutrin, and GS2 are expressed in rat islets and INS832/13 cells, with ATGL being the most abundant. We then focused on the role of ATGL in fuel-stimulated insulin secretion in two models, INS832/13 β-cells in which ATGL expression was reduced by RNA interference-knockdown (ATGL-KD) and ATGL^−/− mice.     

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag⁺ and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag⁺ sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag⁺ sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd²⁺, a membrane-impermeant soft metal ion with properties similar to Ag⁺. Cd²⁺ inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.F₁F₀ ATP synthase utilizes the energy stored in an H⁺ or Na⁺ electrochemical gradient to synthesize ATP in bacteria, mitochondria, and chloroplasts (1–4). The ATP synthase complex is composed of two sectors, i.e. a water-soluble F₁ sector that is bound to a membrane-embedded F₀ sector. In bacteria, F₁ is composed of five subunits in an α₃β₃γδϵ ratio and contains three catalytic sites for ATP synthesis and/or hydrolysis centered at the α-β subunit interfaces. F₀ is composed of three subunits in an a₁b₂c_10–15 ratio and functions as the ion-conducting pathway (5–9). Ion translocation through F₀ drives rotation of a cylindrical ring of c-subunits that is coupled to rotation of the γ subunit within the (αβ)₃ hexamer of F₁ to force conformational changes in the three active sites and in turn drive synthesis of ATP by the binding change mechanism (1–4, 10–13).Subunit c of F₀ folds in the membrane as a hairpin of two extended α-helices. In Escherichia coli, 10 copies of subunit c pack together to form a decameric ring with TMH1² on the inside and TMH2 on the periphery (6, 14). An atomic resolution structure of the Na⁺-translocating c₁₁-ring from Ilyobacter tartaricus was recently published by Meier et al. (8). In the c₁₁ structure, the Na⁺ binding site is formed by two interacting c subunits. The essential Na⁺-binding Glu residue, which corresponds to Asp-61 in E. coli, is located in TMH2 at the middle of the lipid bilayer. Subunit a consists of five transmembrane helices, four of which likely interact as a four-helix bundle (15–18). Subunit a lies on the periphery of the c-ring with TMHs 4 and 5 from subunit a and TMH2 from subunit c forming the a-c interface (18–21). During ion translocation through F₀, the essential Arg-210 on TMH4 of subunit a is postulated to facilitate the protonation/deprotonation cycle at Asp-61 of subunit c and cause the rotation of the c-ring past the stationary subunit a (3, 4, 19).Chemical modification of cysteine-substituted transmembrane proteins has been widely used as a means of probing the aqueous accessible regions (22–24). The reactivity of a substituted cysteine to thiolate-directed probes provides an indication of aqueous accessibility because the reactive thiolate species is preferentially formed in an aqueous environment. The aqueous accessibility of the five TMHs in subunit a of E. coli F₀ has been probed using Ag⁺ and NEM (19, 25–27). The results suggest the presence of an aqueous accessible channel in subunit a in the center of TMHs 2–5 extending from the periplasm to the center of the membrane. Protons entering through this periplasmic access channel are postulated to bind to the essential Asp-61 residues of the c-ring and exit to the cytoplasm by a still uncertain pathway at the peripheral face of aTMH4 with protonation/deprotonation of Asp-61 driving c-ring rotation.During H⁺-driven ATP synthesis, two models for the pathway by which H⁺ or Na⁺ exit to the cytoplasm have been proposed. The first model proposes that the ions bound at Asp-61 exit to the cytoplasm via a half-channel composed at least partially by residues in TMH4 of subunit a (25–27). Chemical modification studies of Cys-substituted subunit a of E. coli revealed an aqueous accessible surface of TMH4 that includes the essential Arg-210 residue, which extended from the center of the membrane to the cytoplasm, suggesting that the ion exit channel may lie at the a-c interface (19, 25). Alternatively studies of the c-ring from the I. tartaricus enzyme indicate that Na⁺ can access Glu-65 in the absence of other F₀ subunits, suggesting an intrinsic channel in subunit c (28, 29). However, no such channel was apparent in the crystal structure of the c₁₁-ring (8). In a previous study (30), we probed the thiolate reactivity of Cys substitutions in the cytoplasmic half of TMH2 in subunit c. These experiments revealed extensive reactivity to sulfhydryl-directed reagents on the peripheral face of cTMH2, supporting the presence of the cytoplasmic exit channel at the a-c interface. In this study, we extended the survey of aqueous accessibility in transmembrane regions by probing thiolate reactivity of Cys substitutions in TMH1 and in the periplasmic half of TMH2. The reactivity of Cys substituted into these regions proved to be more limited. Only a small region of TMH1, lying directly behind Asp-61, was reactive with Ag⁺. In addition to Ag⁺, we used Cd²⁺ as a complementary, membrane-impermeant probe for aqueous accessibility. The survey of Cd²⁺ sensitivity confirmed that aqueous accessibility from the cytoplasm is much greater for residues packing at the periphery of the c-ring. The experiments reported here distinguish the aqueous accessible and inaccessible regions of the c-ring and strengthen evidence that the cytoplasmic H⁺ exit channel is situated at the a-c interface.