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The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design., Serotonin (5-hydroxytryptamine or 5-HT) modulates the activity of the central nervous system as well as processes throughout the body ranging from cardiovascular function to digestion, body temperature, endocrinology and reproduction [...]

Abstract: Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane enzyme that catalyzes the oxidation of sulfide and the reduction of ubiquinone. Ubiquinone binds to a conserved hydrophobic domain and shuttles electrons [...]

The present work is devoted to probing the molecular interaction of N,N',N'-triethylenethiophosphoramide (thioTEPA) with human serum albumin (HSA) using UV-visible and fluorescence spectroscopies. Further, molecular dynamics and molecular docking simulations were used to investigate the binding site of thioTEPA. The outcomes of the spectroscopic observations and also the Stern-Volmer and van't Hoff equations were employed to determine the binding thermodynamic parameters. It was found out that the interaction of thioTEPA with HSA is enthalpy driven through a quenching mechanism that is both static and dynamic at domain I of HSA. No significant changes in the local and overall secondary structure, polarity, and hydrophobicity of HSA were observed. The low values of binding free energies and binding constants are evidence for necessary high dosage of thioTEPA in chemotherapies. These results may be attributed to the spherical geometry and steric hindrance of the free vibrating aziridinyl groups of thioTEPA. Key words: human serum albumin, thioTEPA, fluorescence spectroscopy, molecular dynamics simulation, docking. Resume: Les presents travaux ont pour objectif d'examiner l'interaction moleculaire entre le N,N',N'- triethylenethiophosphoramide (thioTEPA) avec l'albumine serique humaine (HSA) a l'aide de la spectroscopie UV-visible et de la spectroscopie par fluorescence. Nous avons egalement explore le site de liaison du thioTEPA par le biais de la simulation de la dynamique moleculaire et de la simulation interactive de reactions chimiques. Les resultats tires des observations spectroscopiques et des equations de SternVolmer et de van't Hoff ont servi a determiner les parametres thermodynamiques de liaison. Nous avons decouvert que l'interaction entre le thioTEPA et la HSA est gouvernee par l'enthalpie et procede selon un mecanisme d'extinction, tant statique que dynamique, au domaine I du HSA. Nous n'avons observe aucun changement notable de la structure secondaire locale et globale, ni de la polarite ou de l'hydrophobicite de la HSA. Les faibles valeurs des energies libres d'association et des constantes d'association montrent la necessite d'utiliser des doses elevees de thioTEPA en chimiotherapie. Ces resultats pourraient etre attribuables a la geometrie spherique et a l'encombrement sterique des groupements aziridinyles du thioTEPA en oscillation libre. [Traduit par la Redaction] Mots-cles : albumine serique humaine, thioTEPA, spectroscopie par fluorescence, simulation de la dynamique moleculaire, simulation interactive de reactions chimiques., Introduction Plasma proteins play an important role in transportation and deposition of substances such as fatty acids, hormones, and medicinal drugs in the circulatory system. Therefore, it is important to [...]

The suppressor domain of inositol 1,4,5 trisphosphate receptor ([IP.sub.3]R) has critical roles in regulating the calcium channel by interacting with many binding partners. The residue 49-53 (PPKKF) of the suppressor domain was suggested to be a canonical Homer EVH1 domain binding site and is also the first a part of calmodulin (CaM) binding site. As CaM-binding of the suppressor domain has been shown to involve large-scale conformational changes, we studied the binding characteristics of the Homer EVH1-suppressor domain with NMR spectroscopy and biochemical pull-down assays for mutants. Our data show that the suppressor domain employs the PPKKF motif in a similar but subtly different way compared to previously characterized interactions, and that the suppressor domain does not undergo large-scale conformational changes. Chemical shift assignments of the Homer3 EVH1 domain found that a new set of residues, located at the opposite side of the previously reported binding site, is also involved in binding, which was confirmed by mutant binding assays. Further analysis suggests that F40 in the new binding sites may have a critical role as a conformational lock-switch in Homer-target binding. The proposed mechanism is implicated in the signaling network involving calcium channels. Key words: Homer3 EVH1 domain, inositol 1,4,5 trisphosphate receptor, NMR. Le domaine suppresseur du recepteur d'IP3 joue un role critique dans la regulation du canal calcique par son interaction avec plusieurs partenaires de liaison. II a ete suggere que les residus 49-53 (PPKKF) du domaine suppresseur constituent un domaine canonique de liaison EVH1 de Homer, et aussi la premiere partie du domaine de liaison de la calmoduline (CaM). Puisque la liaison de la CaM au domaine suppresseur implique un large spectre de changements de conformation, nous avons etudie les caracteristiques de liaison de EVH1 de Homer au domaine suppresseur par la spectroscopie en RMN et des dosages biochimiques par precipitation de mutants. Nos donnees montrent que le domaine suppresseur utilise le motif PPKKF de maniere similaire mais subtilement differente comparativement aux interactions caracterisees precedemment, et que le domaine suppresseur ne subit pas de changements de conformation importants. Les assignations par deplacement chimique du domaine EVH1 de Homer ont revele qu'un nouvel ensemble de residus, localises a l'oppose du site de liaison precedemment rapporte, sont aussi impliques dans la liaison, ce qui a ete confirme par des tests de liaison de mutants. Une analyse plus approfondie suggere que le residu F40 du nouveau site de liaison puisse jouer un role critique comme interrupteur de blocage dans la liaison ciblant Homer. Le mecanisme propose est implique dans le reseau de signalisation affectant les canaux calciques. [Traduit par la Redaction] Mots-cles: domaine EVH1 de Homer3, recepteur de l'inositol 1,4,5 triphosphate, RMN., Introduction [IP.sub.3]R is a large tetrameric intracellular [Ca.sup.2+]-release channel composed of about 2700 amino acids (Taylor et al. 2004). Its primary sequence can be functionally separated into five sub-domains: the [...]

Retrotransposons have been used frequently for the development of molecular markers by using their insertion polymorphisms among cultivars, because multiple copies of these elements are dispersed throughout the genome and inserted copies are inherited genetically. Although a large number of long terminal repeat (LTR) retrotransposon families exist in the higher eukaryotic genomes, the identification of families that show high insertion polymorphism has been challenging. Here, we performed an efficient screening of these retrotransposon families using an Illumina HiSeq2000 sequencing platform with comprehensive LTR library construction based on the primer binding site (PBS), which is located adjacent to the 5' LTR and has a motif that is universal and conserved among LTR retrotransposon families. The paired-end sequencing library of the fragments containing a large number of LTR sequences and their insertion sites was sequenced for seven strawberry (Fragaria x ananassa Duchesne) cultivars and one diploid wild species (Fragaria vesca L.). Among them, we screened 24 families with a 'unique' insertion site that appeared only in one cultivar and not in any others, assuming that this type of insertion should have occurred quite recently. Finally, we confirmed experimentally the selected LTR families showed high insertion polymorphisms among closely related cultivars. Key words: retrotransposon, primer binding site, high-throughput sequencing, polymorphism, molecular markers. Les retrotransposons ont frequemment ete employes pour developper des marqueurs moleculaires en exploitant le polymorphisme des sites d'insertion entre cultivars, lequel decoule du fait que de multiples copies de ces elements sont dispersees au sein du genome et que ces copies sont transmises de maniere hereditaire. Bien qu'il existe un grand nombre de familles de retrotransposons a longues repetitions terminales (LTR) au sein des genomes des eucaryotes superieurs, il demeure difficile d'identifier les familles qui presentent un grand polymorphisme au niveau des sites d'insertion. Dans ce travail, les auteurs ont efficacement crible ces familles de retrotransposons au moyen du sequencage de librairies de LTR sur une plateforme Illumina HiSeq2000. Ces librairies ont ete preparees en exploitant le site d'appariement de l'amorce (PBS pour << primer binding site >>), une region adjacente au 5' LTR qui comprend un motif universel et conserve parmi les diverses familles de retrotransposons a LTR. Les deux extremites des fragments captures dans ces librairies, lesquelles contenaient un grand nombre de sequences LTR et leurs sites d'insertion, ont ete sequencees chez sept cultivars de fraisiers (Fragaria x ananassa Duschene) et une espece sauvage diploide (Fragaria vesca L.). Parmi ces librairies, les auteurs ont examine 24 familles presentant un site d'insertion << unique >> observe chez un cultivar et aucune autre en postulant que ce type d'insertion serait survenu recemment. Enfin, les auteurs ont confirme experimentalement que les familles de LTR choisies presentaient un fort polymorphisme du site d'insertion parmi des cultivars tres apparentes. [Traduit par la Redaction] Mots-cles: retrotransposons, site d'appariement de l'amorce, sequencage a haut debit, polymorphisme, marqueurs moleculaires., Introduction Retrotransposons are the major component of eukaryotic genomes and have contributed to their evolution and diversification (Kumar and Bennetzen 1999; Feschotte et al. 2002; Wessler 2006). Among them, the [...]

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi) (1,2) and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence (5), whereupon they produce a 3' single-stranded DNA tail onto which they initiate loading of the RecA protein (6). Consequently, regulation of the AddAB/ RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments (7-9)., We have previously determined the crystal structures of initiation complexes of AddAB and RecBCD bound to a DNA end (10,11). We have now crystallized AddAB in a translocation-like state by [...]

This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/ regulation of endothelin-1 (ET-1) to its receptor subtypes A ([ET.sub.A]R) and B ([ET.sub.B]R) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [[sup.125]I]-ET-1 was employed to estimate ET-1 binding affinity (τ = 1/[K.sub.-n]) to [ET.sub.A]R and [ET.sub.B]R at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of [ET.sub.A]R and [ET.sub.B]R were detected using Western blot. No significance differences were detected in the τ of [ET.sub.A]R and [ET.sub.B]R between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the t value for [ET.sub.A]R and [ET.sub.B]R on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, [ET.sub.A]R and [ET.sub.B]R densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated [ET.sub.A]R and down-regulated [ET.sub.B]R band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus. Key words: diabetes mellitus, renin-angiotensin system, endothelin, exendin-4, aliskiren, insulin, binding affinity. Cette etude se concentre sur l'impact de l'aliskiren et (ou) de l'analogue du GLP-1 (glucagon-like peptide 1) sur l'affinite et la regulation de la liaison de l'endotheline-1 (ET-1) a ses sous-types de recepteurs A ([ET.sub.A]R) et B ([ET.sub.B]R), dans l'endothelium coronaire et les cardiomyocytes de rats presentant un diabete de type 1. Sept groupes ont ete utilises : (i) rats normaux, (ii) rats avec le diabete induit, (iii) rats avec le diabete induit traite a l'insuline, (iv) rats avec le diabete induit traite a l'exendin-4, (v) rats avec le diabete induit traite a l'aliskiren, (vi) rats avec le diabete induit traite conjointement a l'insuline et l'aliskiren, et (vii) rats avec le diabete induit traite conjointement a l'exendin-4 et l'aliskiren. L'affinite de la liaison (τ = 1/[K.sub.-n]) a l'[ET.sub.A]Reta l'[ET.sub.B]R dans l'endothelium coronaire et les cardiomyocytes a ete estimee en perfusant le coeur avec du [[sup.125]I]-ET-1. Les niveaux plasmatiques d'ET-1 ont ete mesures par dosage immuno-enzymatique alors que la densite de l'[ET.sub.A]R et l'[ET.sub.B]R a ete estimee par buvardage Western. Aucune difference significative de τ a l'[ET.sub.A]R et l'[ET.sub.B]R de l'endothelium coronaire et des cardiomyocytes n'a ete detectee entre les rats diabetiques et normaux. L'exendin-4 normalisait la valeur de τ de l'[ET.sub.A]R et l'[ET.sub.B]R de l'endothelium coronaire, alors que l'aliskiren la normalisait dans les cardiomyocytes. En outre, les densites d'[ET.sub.A]R et d'[ET.sub.B]R etaient normalisees par les traitements a l'aliskiren et l'exendin-4 utilises seuls, comparativement a une augmentation de la densite de la bande d'[ET.sub.A]R et diminution de celle d'[ET.sub.B]R chez les animaux diabetiques. Nos donnees indiquent que l'aliskiren attenue l'hypertrophie associee au diabete dans le diabete sucre de type 1. [Traduit par la Redaction] Mots-cles: diabete sucre, systeme renine-angiotensine, endotheline, exendin-4, aliskiren, insuline., Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia (Fowler 2008). It is widely associated with public health: 344 million people worldwide have diabetes, and this number is [...]

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for [...]

Pyolysin (PLO) belongs to the homologous family of the cholesterol-dependent cytolysins (CDCs), which bind to cell membranes containing cholesterol to form oligomeric pores of large size. The CDC monomer structure consists of 4 domains. Among these, the C-terminal domain 4 has been implicated in membrane binding of the monomer, while the subsequent processes of oligomerization and membrane insertion have primarily been assigned to other domains of the molecule. Recombinantly expressed or proteolytic fragments that span domain 4 of the CDCs streptolysin O and perfringolysin O bind to membranes but fail to oligomerize, and they inhibit the activity of the respective wild-type toxins. We report here that the isolated domain 4 of pyolysin (PLO-D4) not only binds to membranes but also forms oligomers with itself, as well as hybrid oligomers with the full-length toxin. As expected, the pure PLO-D4 oligomers are devoid of pore-forming activity. Surprisingly, however, within hybrid oligomers, PLO-D4 not only fails to inhibit, but even amplifies the hemolytic activity of the full-length toxin, to an extent similar to that of doubling the amount of the full-length toxin alone. We propose that this amplification may be related to the kinetics of the oligomerization reaction. Overall, our findings indicate a greater role of domain 4 in the oligomerization of CDCs than previously demonstrated. Key words: pyolysin, cholesterol, oligomerization, hemolysis. Resume : La pyolysine (PLO) appartient a la famille homologue des cytolysines dependantes du cholesterol (CDC) qui lient les membranes cellulaires contenant du cholesterol afin de former des pores oligomeriques de grande taille. La structure monomerique de CDC se compose de quatre domaines. Parmi eux, le domaine 4 C-terminal a ete implique dans la liaison du monomere a la membrane, alors que les processus subsequents d'oligomerisation et d'insertion dans la membrane ont ete initialement attribues aux autres domaines de la molecule. Des fragments recombinants ou proteolytiques qui couvrent le domaine 4 de la streptolysine O (SLO) et de la perfringolysine O (PFO) se lient aux membranes mais ne reussissent pas a s'oligomeriser, et ils inhibent l'activite des toxines sauvages correspondantes. Nous rapportons ici que le domaine 4 de la pyolysine (PLO-D4) isole se lie non seulement aux membranes mais forme aussi des oligomeres avec lui-meme, ainsi que des oligomeres hybrides avec la toxine de pleine longueur. Comme prevu, les oligomeres constitues uniquement de PLO-D4 ne forment pas de pore. Etonnamment toutefois, a l'interieur des oligomeres hybrides, non seulement le PLO-D4 ne parvient- il pas a inhiber l'activite hemolytique de la toxine de pleine longueur, mais il l'amplifie meme a un niveau similaire a celui obtenu si l'on doublait la quantite de toxine de pleine longueur seule. Nous proposons que cette amplification puisse etre reliee a la cinetique de la reaction d'oligomerisation. Dans l'ensemble, nos donnees indiquent que le domaine 4 joue un role plus important dans l'oligomerisation des CDC que prevu initialement. Mots-cles: pyolysine, cholesterol, oligomerisation, hemolyse., Introduction The cholesterol-dependent cytolysins (CDCs) are a family of homologous pore-forming toxins produced by various species of Gram-positive bacteria (Heuck et al. 2010). Among its members, streptolysin O (SLO), perfringolysin [...]

Acetals are molecular substructures that contain two oxygen-carbon single bonds at the same carbon atom, and are used in cells to construct carbohydrates and numerous other molecules. A distinctive subgroup are spiroacetals, acetals joining two rings, which occur in a broad range of biologically active compounds, including small insect pheromones and more complex macrocycles (1,2). Despite numerous methods for the catalytic asymmetric formation of other commonly occurring stereocentres, there are few approaches that exclusively target the chiral acetal centre and none for spiroacetals (3,4). Here we report the design and synthesis of confined Bronsted acids based on a [C.sub.2]-symmetric imidodiphosphoric acid motif, enabling a catalytic enantioselective spiroacetalization reaction. These rationally constructed Bronsted acids possess an extremely sterically demanding chiral microenvironment, with a single catalytically relevant and geometrically constrained bifunctional active site. Our catalyst design is expected to be of broad utility in catalytic asymmetric reactions involving small and structurally or functionally unbiased substrates., Spiroacetal natural products are widely found in insects, plants, and bacterial and marine sources, and display a diverse set of biological activities (1,2). Their spiroacetal subunit is not only essential [...]

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2- directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-- with PG9--involves a site of vulnerability comprising just two glycans and a strand., As the sole viral target of neutralizing antibodies, the HIV-1 viral spike has evolved to evade antibody-mediated neutralization (reviewed in ref. 1). V1/V2 of the gp120 component of the viral [...]

UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 [Angstrom] resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-[Angstrom]-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p's C-terminal region as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 [Angstrom] thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments. myosin stability | processivity | chaperone doi/ 10.1073/pnas.1013038107

A theoretical framework is presented to clarify the molecular determinants of ion selectivity in protein binding sites. The relative free energy of a bound ion is expressed in terms of the main coordinating ligands coupled to an effective potential of mean force representing the influence of the rest of the protein. The latter is separated into two main contributions. The first includes all the forces keeping the ion and the coordinating ligands confined to a microscopic subvolume but does not prevent the ligands from adapting to a smaller or larger ion. The second regroups all the remaining forces that control the precise geometry of the coordinating ligands best adapted to a given ion. The theoretical framework makes it possible to delineate two important limiting cases. In the limit where the geometric forces are dominant (rigid binding site), ion selectivity is controlled by the ion-ligand interactions within the matching cavity size according to the familiar 'snug-fit' mechanism of host-guest chemistry. In the limit where the geometric forces are negligible, the ion and ligands behave as a 'confined microdroplet' that is free to fluctuate and adapt to ions of different sizes. In this case, ion selectivity is set by the interplay between ion-ligand and ligand-ligand interactions and is controlled by the number and the chemical type of ion-coordinating ligands. The framework is illustrated by considering the ion-selective binding sites in the KcsA channel and the LeuT transporter. computations | free energy | ion coordination | KcsA | LeuT doi/ 10.1073/pnas.1007150107

BK-type [K.sup.+] channels are activated by voltage and intracellular [Ca.sup.2+], which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different [Ca.sup.2+] binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this [Ca.sup.2+] binding site. This site is specific for binding to [Ca.sup.2+] but not [Cd.sup.2+]. Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of [Ca.sup.2+] by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how [Ca.sup.2+] binding at this site opens the activation gate of the channel that is remotely located in the membrane. [Ca.sup.2+]-activated | allosteric gating | [Ca.sup.2+] binding site | [Cd.sup.2+] | Slo1 doi/ 10.1073/pnas.1010124107

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family 'zyklopen.' J. Nutr. 140: 1728-1735, 2010. doi: 10.3945/jn.109.117531.

Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioll a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis. DOI 10.1099/mic.0.035758-0

Polyamine block of inwardly rectifying potassium (Kir) channels underlies their steep voltage dependence observed in vivo. We have examined the potency, voltage dependence, and kinetics of spermine block in dimeric Kir2.1 constructs containing one nonreactive subunit and one cysteine-substituted subunit before and after modification by methanethiosulfonate (MTS) reagents. At position 169C (between the D172 'rectification controller' and the selectivity filter), modification by either 2-aminoethyl MTS (MTSEA) or 2-(trimethylammonium)ethyl MTS (MTSET) reduced the potency and voltage dependence of spermine block, consistent with this position overlapping the spermine binding site. At position 176C (between D172 and the M2 helix bundle crossing), modification by MTSEA also weakened spermine block. In contrast, MTSET modification of 176C dramatically slowed the kinetics of spermine unblock, with almost no effect on potency or voltage dependence. The data are consistent with MTSET modification of 176C introducing a localized barrier in the inner cavity, resulting in slower spermine entry into and exit from a 'deep' binding site (likely between the D172 rectification controller and the selectivity filter), but leaving the spermine binding site mostly unaffected. These findings constrain the location of deep spermine binding that underlies steeply voltage-dependent block, and further suggest important chemical details of high affinity binding of spermine in Kir2.1 channels--the archetypal model of strong inward rectification. doi/ 10.1085/jgp.200910253

The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like [beta]-sandwich fold with two extra helix-turn-helix inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAR In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP. doi/ 10.1073/pnas.1000293107 Hippo pathway | oncogene | crystallography

Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90[degrees] helical bend Iocalized to domain Ila that positions the apical Ioop of domain IIb of the IRES near the 40S ribosomal E-site to promote elF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain Ila construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol, Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class. antiviral | hepatitis C virus | molecular dynamics | RNA structure doi /10.1073/pnas.0911896107

DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells. DOI 10.1099/mic.0.027144-0

Dynamic solvation at binding and active sites is critical to protein recognition and enzyme catalysis. We report here the complete characterization of ultrafast solvation dynamics at the recognition site of photoantenna molecule and at the active site of cofactor/ substrate in enzyme photolyase by examining femtosecond-resolved fluorescence dynamics and the entire emission spectra. With direct use of intrinsic antenna and cofactor chromophores, we observed the local environment relaxation on the time scales from a few picoseconds to nearly a nanosecond. Unlike conventional solvation where the Stokes shift is apparent, we observed obvious spectral shape changes with the minor, small, and large spectral shifts in three function sites. These emission profile changes directly reflect the modulation of chromophore's excited states by locally constrained protein and trapped-water collective motions. Such heterogeneous dynamics continuously tune local configurations to optimize photolyase's function through resonance energy transfer from the antenna to the cofactor for energy efficiency and then electron transfer between the cofactor and the substrate for repair of damaged DNA. Such unusual solvation and synergetic dynamics should be general in function sites of proteins. function-site solvation | ultrafast dynamics | spectral tuning | protein rigidity and flexibility | femtosecond-resolved emission spectra www.pnas.org/cgi/doi/10.1073/pnas.1000001107

Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase complex that recognizes Nrf2, and also acts as a cellular sensor for xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor regulating the expression of cytoprotective enzyme genes in response to such stresses. Under unstressed conditions Keap1 binds Nrf2 and results in rapid degradation of Nrf2 through the proteasome pathway. In contrast, upon exposure to oxidative and electrophilic stress, reactive cysteine residues in intervening region (IVR) and Broad complex, Tramtrack, and Bric-a-Brac domains of Keap1 are modified by electrophiles. This modification prevents Nrf2 from rapid degradation and induces Nrf2 activity by repression of Keap1. Here we report the structure of mouse Keap1 homodimer by single particle electron microscopy. Three-dimensional reconstruction at 24-[Angstrom] resolution revealed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob. Each sphere has a tunnel corresponding to the central hole of the [beta]-propeller domain, as determined by x-ray crystallography. The IVR domain appears to surround the core of the [beta]-propeller domain. The unexpected proximity of IVR to the [beta]-propeller domain suggests that any distortions generated during modification of reactive cysteine residues in the IVR domain may send a derepression signal to the [beta]-propeller domain and thereby stabilize Nrf2. This study thus provides a structural basis for the two-site binding and hinge-latch model of stress sensing by the Nrf2-Keap1 system. www.pnas.org/cgi/doi/10.1073/pnas.0914036107

We have taken a synthetic biology approach to the generation and screening of transcription factor binding sites for activity in human cells. All possible 10-met DNA sequences were printed on microarrays as 100-mers containing 10 repeats of the same sequence in tandem, yielding an oligonucleotide library of 52,429 unique sequences. This library of potential enhancers was introduced into a retroviral vector and screened in multiple cell lines for the ability to activate GFP transcription from a minimal CMV promoter. With this method, we isolated 100 bp synthetic enhancer elements that were as potent at activating transcription as the WT CMV immediate early enhancer. The activity of the recovered elements was strongly dependent on the cell line in which they were recovered. None of the elements were capable of achieving the same levels of transcriptional enhancement across all tested cell lines as the CMV enhancer. A second screen, for enhancers capable of synergizing with the elements from the original screen, yielded compound enhancers that were capable of twofold greater enhancement activity than the CMV enhancer, with higher levels of activity than the original synthetic enhancer across multiple cell lines. These findings suggest that the 10-mer synthetic enhancer space is sufficiently rich to allow the creation of synthetic promoters of all strengths in most, if not all, cell types. Transcription | GFP reporter | RNA interference | synthetic biology www.pnas.org/cgi/doi/10.1073/pnas.0914803107

Salmonella plasmid pFPTB1 includes a Tn3-1ike transposon and a Xer recombination site, fpr, which mediates site-specific recombination at efficiencies lower than those required for stabilizing a plasmid by dimer resolution. Mutagenesis and comparative studies with mwr, a site closely related to fpr, indicate that there is an interdependence of the sequences in the XerC binding region and the central region in Xer site-specific recombination sites. doi: 10.1128/JB.01082-09

Using Autodock, docking of penicillin G to the crystal structures of penicillin-recognizing enzymes leads to an alignment in the active site Ser-X-X-Lys region consisting of the serine hydroxyl group, the terminal amino group of lysine, a second hydroxyl group, and the N-C=O of the β-lactam. This alignment is consistent with the notion that acylation of the serine hydroxyl group proceeds by a one-step cooperative mechanism in which C-O bond formation and proton transfer to the β-lactam nitrogen take place through a heteroatom bridge. For the cooperative ring opening of penam by two molecules of methanol and one molecule of methylamine or one molecule of water, density functional theory with the B3LYP DFT gradient-corrected functional and the 6-31G(d) basis set reproduces the alignment seen in the docked structures. Methylamine lowers the barrier calculated at MP2/6-31G(d) from the DFT-optimized geometries by 3 kcal/mol; water increases the barrier by 4 kcal/mol. The function of the conserved lysine in the active sites of penicillin- recognizing enzymes is therefore to catalyze the formation of an acyl enzyme by a cooperative mechanism. Key words: Autodock, cooperative mechanism, density functional theory (DFT), acyl enzyme. Les simulations a l'aide du programme Autodock montrent que l'arrimage de la penicilline G aux structures cristallines des enzymes reconnaissant la penicilline conduit a un alignement dans la region du site actif Ser-X-X-Lys forme du groupe hydroxyle de la lysine, du groupe amine terminal de la lysine, du second groupe hydroxyle et du N-C=O du β- lactame. Cet arrangement est en accord avec la notion selon laquelle l'acylation du groupe hydroxyle de la lysine se produit par un mecanisme cooperatif en une etape dans lequel la formation de la liaison GO et le transfert de proton a partir de l'azote du β-lacam se produit par un pont heteroatomique. Pour l'ouverture de cycle cooperative du penam par deux molecules de methanol et une molecule de methylamine on une molecule d'eau, l'utilisation de la theorie de la fonctionnelle de la densite, TFD, avec une fonctionnelle corrigee pour le gradient, B3LYP, et un ensemble de base 6-31G(d) permet de reproduire l'alignement observe dans les arrimages de structures. Pour la methylamine la barriere calculee an niveau MP2/6-31G(d) est abaissee de 3 kcal/mol par rapport a la valeur obtenue par la TFD avec des geometries optimisees. Mots-cles : Autodock, mecanisme cooperatif, theorie de la fonctionnelle de la densite (TFD), enzyme acyle. [Traduit par la Redaction], Penicillin-binding proteins (PBPs), the targets of β-lactam antibiotics, (1) and β-lactamases, which defend bacteria from the action of β-lactam antibiotics, (2) are related to each other structurally, (3) kinetically, (4) [...]

DevR activates the transcription of ~48 genes in response to hypoxia and other stresses and triggers metabolic downshift and dormancy development in Mycobacterium tuberculosis, tgs1 and Rv3131 encode triacyl-glycerol synthase and a putative nitroreductase, respectively, and both are members of the DevR regulon. This study aimed to understand how a single putative DevR binding site identified previously could sustain powerful induction of divergent tgs1-Rv3131 genes. DNase I footprinting revealed that phosphorylated DevR in fact binds to two sites symmetrically located at -42.5 and -63.5 bp from transcription start points of both genes. DevR first bound to the high-affinity site, P, and cooperatively recruited another DevR molecule to the secondary low-affinity site, S, to activate tgs1-Rv3131 transcription by ~210- and ~ll0-fold, respectively. The presence of a single P site significantly reduced activation of tgs1 expression and abolished Rv3131 activity, reinforcing the requirement of two binding sites for robust expression in both directions. P site inversion abolished tgs1 but not Rv3131 transcription despite DevR occupancy at both sites. The lack of tgs1 expression is most likely due to disruption of its -35 promoter element rather than inversion of the binding site per se. We conclude that (i) an overlap of a DevR binding site and -35 sequence is indispensable for promoter activation, (ii) DevR interaction with two binding sites is obligatory for synergistic activation of tgs1-Rv3131 promoters, and (iii) DevR interaction with binding sites of different affinities offers scope for temporal and differential expression of target genes. doi: 10.1128/JB.00310-09

Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine-Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins. glycobiology | influenza | nidovirus | sialate-O-acetylesterase | X-ray crystallography

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding [Ca.sup.2+] and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the [Ca.sup.2+] locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed. actin | calcium activated | calcium dependent | TIRF

The dendritic cell receptor DC-SIGN mediates pathogen recognition by binding to glycans characteristic of pathogen surfaces, including those found on HIV. Clustering of carbohydrate-binding sites in the receptor tetramer is believed to be critical for targeting of pathogen glycans, but the arrangement of these sites remains poorly understood. Surface force measurements between apposed lipid bilayers displaying the extracellular domain of DC-SIGN and a neoglycolipid bearing an oligosaccharide ligand provide evidence that the receptor is in an extended conformation and that glycan docking is associated with a conformational change that repositions the carbohydrate-recognition domains during ligand binding. The results further show that the lateral mobility of membrane-bound ligands enhances the engagement of multiple carbohydrate-recognition domains in the receptor oligomer with appropriately spaced ligands. These studies highlight differences between pathogen targeting by DC-SIGN and receptors in which binding sites at fixed spacing bind to simple molecular patterns. adhesion | molecular recognition | pathogen selectivity | multivalent receptors

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an [IC.sub.50] of 1.2 [+ or -] 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 [micro]M gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1-Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general. ciguatera | neurotoxin site 5 | polycyclic ether toxin | potassium channels | Kv3.1

FtsE and FtsX of Escherichia coli constitute an apparent ABC transporter that localizes to the septal ring. In the absence of FtsEX, cells divide poorly and several membrane proteins essential for cell division are largely absent from the septal ring, including FtsK, FtsQ, FtsI, and FtsN. These observations, together with the fact that ftsE and ftsX are cotranscribed with ftsY, which helps to target some proteins for insertion into the cytoplasmic membrane, suggested that FtsEX might contribute to insertion of division proteins into the membrane. Here we show that this hypothesis is probably wrong, because cells depleted of FtsEX had normal amounts of FtsK, FtsQ, FtsI, and FtsN in the membrane fraction. We also show that FtsX localizes to septal rings in cells that lack FtsE, arguing that FtsX targets the FtsEX complex to the ring. Nevertheless, both proteins had to be present to recruit further Fts proteins to the ring. Mutant FtsE proteins with lesions in the ATP-binding site supported septal ring assembly (when produced together with FtsX), but these rings constricted poorly. This finding implies that FtsEX uses ATP to facilitate constriction rather than assembly of the septal ring. Finally, topology analysis revealed that FtsX has only four transmembrane segments, none of which contains a charged amino acid. This structure is not what one would expect of a substrate-specific transmembrane channel, leading us to suggest that FtsEX is not really a transporter even though it probably has to hydrolyze ATP to support cell division.

The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallei [beta]-strands in this fold are presumed to insert into the OF operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The [O.sub.F] locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully bound operator is cooperatively coated by up to eight dimers. [O.sub.F] was subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to D individually are bound with different affinities by ParG (C > A [approximately equal to] B >> D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The [O.sub.F] operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.