Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the “5 + 5 inverted repeat” fold found in other families like the Na + -coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg + -bound occluded. However, the role of Arg + during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg + -bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg + -guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine. Purified detergent-solubilized AdiC particles were dimeric. Reconstitution experiments yielded twodimensional crystals of AdiC-W293L diffracting beyond 6 Å resolution from which we determined the projection structure at 6.5 Å resolution. The projection map showed 10 -12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning ␣-helix. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution.Enteric pathogens such as Shigella, Salmonella, Yersinia, spp., and certain Escherichia coli strains can survive the extremely acidic conditions of the human stomach and cause intestinal diseases (1). To overcome the protective barrier of the gastric acidity, pathogenic and nonpathogenic strains of E. coli have developed acid resistance systems. One of these systems requires arginine to protect E. coli during low pH exposure. This arginine system is composed of an arginine-agmatine exchange transporter and of an acid-activated arginine decarboxylase (2). Acidification of the cytosol is prevented by the consumption of protons through decarboxylation of arginine to agmatine and carbon dioxide. Agmatine is then exported out of the cytosol, and new arginine is imported through the arginineagmatine transporter in a one-to-one exchange stoichiometry (2). This recently identified transport protein is the product of the adiC gene (3, 4). In vitro, AdiC-mediated exchange transport of arginine and agmatine is tightly coupled, electrogenic, and acid-activated (5). AdiC forms stable homodimers in detergent and phospholipid membranes as determined by gel filtration and glutaraldehyde cross-linking experiments (5).The origin of AdiC is somehow controversial as it was assigned to two families of transport proteins, i.e. the amino acid/polyamine/organocation (APC) 7 transporter superfamily (6) and the major facilitator superfamily (MFS) (5, 7). The APC superfamily of transporters consists of nearly 250 members that function as solute-cation symporters and solute-solute antiporters (6). According to hydropathy profile analysis and biochemically established topological features of most prokaryotic and eukaryotic APC superfamily members, both the N and C termini of ...

Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of -crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate-and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in -crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis.

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Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP min؊1 ) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.Coenzyme specificity is an important property of NAD(P)-dependent oxidoreductases that is linked to their metabolic function. Thus the type of coenzyme, NAD ϩ or NADP ϩ , often distinguishes between enzymes involved in alternative pathways (e.g. oxidative versus reductive or degradative versus biosynthetic). Because NAD ϩ and NADP ϩ only differ structurally in the phosphate group esterified at the 2Ј position of adenosine ribose, dehydrogenases must possess a limited number of residues to discriminate between the two coenzyme types. Moreover, among dehydrogenases from a given enzyme family, the same protein fold is often used to bind either coenzyme type and even some enzymes show dual activity, meaning that they can use both coenzymes with similar efficiency (1).A rather unique NADP-dependent alcohol dehydrogenase (ADH8) 1 was discovered in the gastric tissues of amphibians (2). ADH8 belongs to the medium chain dehydrogenase/reductase (MDR) superfamily and is phylogenetically related to the NAD-dependent vertebrate ADH family. This enzyme is active with ethanol and functionally may participate in the reduction of retinal to retinol (k cat /K m all-trans-retinal ϭ 33,750 mM Ϫ1 min Ϫ1 ). Recently, the three-dimensional structure of the ADH8-NADP ϩ binary complex was determined at 1. define a binding pocket for the terminal phosphate group of NADP(H).Henceforth residue numbering will correspond to that of horse ADH1 with the Swiss Prot entry P00327. Interestingly, NADPdependent ADHs from distantly related microorganisms (5-7), also have a glycine and two more hydrophilic residues at the positions corresponding to 223, 224, and 225, respectively. In ADHs, residue 223 is located at the C-terminal end of the second ␤-strand of the Rossmann fold (8) and classically is considered as determinant for coenzyme specificity. The substitution D223G, as found in ADH8, would avoid the possible steric and electrostatic hindrances because of the extra phosphate group of NADP(H). In fact, different attempts to switch the coenzyme specificity in medium chain ADHs have been focused on mutations involving residue 223 (9 -12). However, full reversal of coenzyme specificity, in terms of having a mutant enzyme as catalytically efficient as the wild type, has been rarely achieved. This implies that conversion of coenzyme specificity may require multiple substitutions in the coenzymebinding domain. Other residues found in ADH8, such as Thr 224 and His 225 , which are making hydrogen bonds with the oxygen atoms from the terminal phosphate group (3), could also be important in defining co...

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Different crystal forms diffracting to high resolution have been obtained for two NADP(H)-dependent alcohol dehydrogenases, members of the medium-chain dehydrogenase/reductase superfamily: ScADHVI from Saccharomyces cerevisiae and ADH8 from Rana perezi. ScADHVI is a broad-speci®city enzyme, with a sequence identity lower than 25% with respect to all other ADHs of known structure. The best crystals of ScADHVI diffracted beyond 2.8 A Ê resolution and belonged to the trigonal space group P3 1 21 (or to its enantiomorph P3 2 21), with unit-cell parameters a = b = 102.2, c = 149.7 A Ê , = 120 . These crystals were produced by the hangingdrop vapour-diffusion method using ammonium sulfate as precipitant. Packing considerations together with the self-rotation function and the native Patterson map seem to indicate the presence of only one subunit per asymmetric unit, with a volume solvent content of about 80%. ADH8 from R. perezi is the only NADP(H)-dependent ADH from vertebrates characterized to date. Crystals of ADH8 obtained both in the absence and in the presence of NADP + using polyethylene glycol and lithium sulfate as precipitants diffracted to 2.2 and 1.8 A Ê , respectively, using synchrotron radiation. These crystals were isomorphous, space group C2, with approximate unitcell parameters a = 122, b = 79, c = 91 A Ê , = 113 and contain one dimer per asymmetric unit, with a volume solvent content of about 50%.

Microsymposia C186lysozyme and truncated the N-terminal 19 residues. The structure was determined at the 3.1 Å resolution with a first-generation antihistamine, doxepin. The structure allows us to characterize its ligand-binding pocket in detail. Doxepin sits much deeper in the pocket than the antagonists in other aminergenic G protein coupled receptor (GPCR) structures and directly interacts with the highly conserved Trp428, a key residue in GPCR activation. Asp107, a strictly conserved residue in aminergic receptors, forms an anchor salt bridge with the amine moiety of doxepin. The antihistamine is also surrounded by highly conserved residues among aminergenic receptors including Ile115, Phe424 and Phe432. The well-conserved pocket and its mostly hydrophobic nature contribute to low selectivity of doxepin and other first-generation compounds causing considerable side effects. The pocket is associated with an anion-binding region occupied by a phosphate molecule.Docking of various second-generation antihistamines reveals that the unique carboxyl-group present in this class of compounds interacts with Lys191 and/or Lys179, both of which form part of the anionbinding region and are not conserved in other aminergenic receptors.The structural details of the antihistamine-binding pocket of H1R will be highly beneficial for guiding rational design of new antihistamines that do not penetrate the BBB while maintaining H1R selectivity. The proton dependent oligopeptide transporters (POTs) are a large family of integral membrane proteins that use the inwardly directed proton electrochemical gradient to transport small peptides, amino acids and nitrate across cellular membranes in both pro-and eukaryotic cells. Evolutionarily the POT family sits within the much larger Major Facilitator Superfamily (MFS), members of which contain a common structural motif of 12 transmembrane-spanning alpha-helical segments. The human genome contains four members of this family, two of which, PepT1 and PepT2 are responsible for the absorption of dietary peptides in the small intestine and peptide re-absorption in the kidney. Peptide transporters also contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, such as the beta-lactam antibiotics. To gain further insight into the molecular mechanism of drug and peptide transport, we determined the crystal structure of a prokaryotic member of the POT family, PepT So , with similar substrate specificity and a high degree of sequence conservation to the mammalian PepT proteins [1]. The structure of PepT So , together with our associated kinetic data, provides valuable new insights into mammalian peptide transport and provides the starting point for further structural and biochemical studies on this pharmaceutically important transporter family. Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer and aminoacidurias. The bacterial L-arginine/agmatine antiport...