Putative Active States of a Prototypic G-Protein-Coupled Receptor from Biased Molecular Dynamics

A major current focus of structural work on G-protein-coupled receptors (GPCRs) pertains to the investigation of their active states. However, for virtually all GPCRs, active agonist-bound intermediate states have been difficult to characterize experimentally owing to their higher conformational flexibility, and thus intrinsic instability, as compared to inactive inverse agonist-bound states. In this work, we explored possible activation pathways of the prototypic GPCR bovine rhodopsin by means of biased molecular dynamics simulations. Specifically, we used an explicit atomistic representation of the receptor and its environment, and sampled the conformational transition from the crystal structure of a photoactivated deprotonated state of rhodopsin to the low pH crystal structure of opsin in the presence of 11-trans-retinal, using adiabatic biased molecular dynamics simulations. We then reconstructed the system free-energy landscape along the predetermined transition trajectories using a path collective variable approach based on metadynamics. Our results suggest that the two experimental endpoints of rhodopsin/opsin are connected by at least two different pathways, and that the conformational transition is populated by at least four metastable states of the receptor, characterized by a different amplitude of the outward movement of transmembrane helix 6.     

Comparative Structural and Molecular Characterization of Ribitol-5-Phosphate-Containing Streptococcus oralis Coaggregation Receptor Polysaccharides

The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfβ1-6GalNAcβ1-3Galα1-2ribitol-5-PO₄⁻-6Galfβ1-3Galβ1]_n, thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is α1-2 in strain SK144 versus α1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.The coaggregations observed between different viridans group streptococci and Actinomyces naeslundii (6) provided early evidence for the role of interbacterial adhesion in dental plaque biofilm formation. Interactions between these bacteria were subsequently attributed to binding of A. naeslundii type 2 fimbriae to specific Gal and GalNAc-containing cell wall polysaccharides, referred to as receptor polysaccharides (RPS), on strains of Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii (7, 9, 14). These streptococci inhabit the tooth surface (23), where they grow in close association with type 2 fimbriated A. naeslundii (26) and other members of the dental plaque biofilm community. Growth and biofilm formation were not observed in flow cells when coaggregating strains of S. oralis and A. naeslundii were cultured separately in dilute saliva (27). However, when cultured together, the two strains grew as a mixed-species community, thereby supporting a recognition role for cell surface RPS in biofilm development.Six structural types of RPS have been identified by high resolution nuclear magnetic resonance (NMR) of the cell wall polysaccharides isolated from over 20 coaggregating strains of S. sanguinis, S. gordonii, and S. oralis (8). These polysaccharides are composed of structurally distinct repeating units that contain conserved Galf linked β1-6 to a host-like recognition motif, which is GalNAcβ1-3Gal (Gn) in certain types of RPS and Galβ1-3GalNAc (G) in others. The flexible β1-6 linkage from Galf (34) is thought to function as a hinge, exposing the adjacent host-like motif for adhesin-mediated recognition (21). Whereas both Gn and G types of RPS are recognized by type 2 fimbriated A. naeslundii, only Gn types are recognized by the GalNAc-binding adhesins present on non-RPS-bearing strains of S. sanguinis and S. gordonii (8). Conversely, only G types are coaggregation receptors of certain Veillonella spp. (25). The host-like features of these polysaccharides, although critical for interbacterial adhesion, contribute little to RPS serotype specificity, which instead reflects the immunogenic features of these molecules (21). As a result, the identification of RPS-bearing streptococci requires both serotyping (i.e., serotypes 1, 2, 3, 4, or 5) and receptor typing (i.e., types Gn or G) of these bacteria.A possible molecular approach for the identification of these bacteria is evident from comparative studies of the chromosomal loci (rps) for RPS biosynthesis in different strains (33, 35-37). In this regard, the genes wchA and wchF, which were first identified in Streptococcus pneumoniae (5, 15), encode the first two transferases for synthesis of RPS serotypes 1, 2, and 3. WchA transfers Glc-1-phosphate from UDP-Glc to a carrier lipid, and WchF adds Rha β1-4 to Glc. Subsequent synthesis of both the antigenic and receptor regions in these polysaccharides depends on other encoded transferases (35-37), many of which are distinguishable from those identified in S. pneumoniae. In addition to Glc- and Rha-containing types of RPS, other types have been described that lack these sugars but contain ribitol-5-phosphate (3), in addition to GalNAc, Galp, and Galf, which are common constituents of all types. The ribitol-5-phosphate-containing group, represented by type 4Gn RPS of S. oralis C104 and type 5Gn RPS of S. oralis SK144, is the subject of the present study. The results define the structural and genetic basis of the antigenic difference noted between these polysaccharides. They also reveal a close molecular relationship between these types of RPS and certain capsular polysaccharides (CPS) of S. pneumoniae, most notably those in CPS serogroup 10.     

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

Background

Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.

Methodology

Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4°C, −20°C & −80°C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.

Conclusion

Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.     

牛1gG高亲和力受体的分子克隆及序列分析

报道编码牛 Ig G高亲和力受体 ( bovine Ig G Fc receptor I,bo FcγR )的全长序列 .从牛肺巨噬细胞 c DNA文库中克隆的该片段全长 1 .4kb,其中的 ORF为 1 0 50 bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的 349个氨基酸 ,含有 5个潜在的 N-连接糖基化位点 .与人和鼠的 Ig G高亲和力受体 ( hu FcγR 和 mo FcγR )相比 ,其核苷酸同源性分别为 80 %和 69% ,氨基酸同源性分别为 66%和 55% .研究表明 ,人、牛和鼠的 3种 Ig G高亲和力受体的单体 Ig G结合域高度保守     

牛IgG高亲和力受体的分子克隆及序列分析

报道编码牛IgG高亲和力受体(bovineIgGFcreceptorI,boFcγRI)的全长序列.从牛肺巨噬细胞cDNA文库中克隆的该片段全长1.4kb,其中的ORF为1050bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的349个氨基酸,含有5个潜在的N-连接糖基化位点.与人和鼠的IgG高亲和力受体(huFcγRI和mohγRI)相比,其核苷酸同源性分别为80%和69%,氨基酸同源性分别为66%和55%.研究表明,人、牛和鼠的3种IgG高亲和力受体的单体IgG结合域高度保守.     

牛1gG高亲和力受体的分子克隆及序列分析

报道编码牛 Ig G高亲和力受体 ( bovine Ig G Fc receptor I,bo FcγR )的全长序列 .从牛肺巨噬细胞 c DNA文库中克隆的该片段全长 1 .4kb,其中的 ORF为 1 0 50 bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的 349个氨基酸 ,含有 5个潜在的 N-连接糖基化位点 .与人和鼠的 Ig G高亲和力受体 ( hu FcγR 和 mo FcγR )相比 ,其核苷酸同源性分别为 80 %和 69% ,氨基酸同源性分别为 66%和 55% .研究表明 ,人、牛和鼠的 3种 Ig G高亲和力受体的单体 Ig G结合域高度保守     

Comparative Analyses of Three-Dimensional Models of Bacterial Reaction Centers

Despite the fact that the three-dimensional structure of an integral membrane protein was first determined 20 years ago, structures have been solved for very few membrane proteins. The reaction center is an exception with many mutant and modified structures available from 3 different bacterial species. In order to relate these structures to the function of the reaction center, an accurate assessment of the reliability of the structural models is required. Here we describe the quality of the structures of the bacterial reaction center based upon different criteria, such as evaluation of the geometry of the models and comparison of different models. Overall, the structures are found to be most accurate in the membrane-embedded regions with the periplasmic and cytoplasmic exposed regions having more disorder and differences among the structural models. In general, the cofactors and the surrounding protein regions are among the most accurately determined regions of the protein, except for the secondary quinone and its binding pocket that shows a large variation among structures. The limited accuracy of the secondary quinone is due to its partial occupancy as a consequence of its functional role and to the presence of surface features, including lipids and detergent molecules.     

Stereochemical Preferences Modulate Affinity and Selectivity among Five PDZ Domains that Bind CFTR: Comparative Structural and Sequence Analyses

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Identification of Brevibacteriaceae by Multilocus Sequence Typing and Comparative Genomic Hybridization Analyses

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.Brevibacteriaceae play a major part in the cheese smear community (6, 11). The classification and typing of cheese-related Brevibacteriaceae have been based mainly on molecular methods such as amplified ribosomal DNA restriction enzyme analysis, pulsed-field gel electrophoresis, and ribotyping (8, 10, 12). Recently, the original Brevibacterium linens group was split into two species on the basis of their physiological and biochemical characteristics, the sugar and polyol composition of their teichoic acids, and their 16S rRNA sequence and DNA-DNA hybridization levels. One species remains B. linens and is represented by type strain ATCC 9172. The other, represented by type strain ATCC 9175, has been renamed Brevibacterium aurantiacum. Regarding this new classification, the taxonomic position of cheese-related isolates has to be revisited and potential relationships between phylogenetic affiliation and the potential occurrence of given metabolic characteristics redefined (7). The unfinished genome sequence of B. aurantiacum ATCC 9174 has recently been released by the Joint Genome Institute (http://genome.jgi-psf.org/draft_microbes/breli/breli.home.html). The development of focused phylogenetic approaches using multiple markers in conjunction with whole-genome screening techniques such as comparative genomic hybridization (CGH) has proven to be useful for the detailed characterization of pathogenic species, including food pathogens (3, 5, 9). However, only a few technological species have been investigated at an intraspecies level (2). Our intention was thus to develop modern tools to facilitate the typing of strains of technological interest, for which Brevibacteriaceae could be used as a case study.     

The Pilin Protein FimP from Actinomyces oris: Crystal Structure and Sequence Analyses

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.     

Hemagglutinin Receptor Specificity and Structural Analyses of Respiratory Droplet-Transmissible H5N1 Viruses

Two ferret-adapted H5N1 viruses capable of respiratory droplet transmission have been reported with mutations in the hemagglutinin receptor-binding site and stalk domains. Glycan microarray analysis reveals that both viruses exhibit a strong shift toward binding to “human-type” α2-6 sialosides but with notable differences in fine specificity. Crystal structure analysis further shows that the stalk mutation causes no obvious perturbation of the receptor-binding pocket, consistent with its impact on hemagglutinin stability without affecting receptor specificity.     

Dual Activation of the Bile Acid Nuclear Receptor FXR and G-Protein-Coupled Receptor TGR5 Protects Mice against Atherosclerosis

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE^−/− mice and LDLR^−/− mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE^−/− and LDLR^−/− mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE^−/− mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.     

Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA

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Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase

Aldo-keto reductases (AKRs) are a large superfamily of NADPH-dependent enzymes that catalyze the reduction of aldehydes, aldoses, dicarbonyls, steroids, and monosaccharides. While their precise physiological role is generally unknown, AKRs are nevertheless involved in the detoxification of a broad range of toxic metabolites. Mycobacteria contain a number of AKRs, the majority of which are uncharacterised. Here, we report the 1.9 and 1.6 Å resolution structures of the apoenzyme and NADPH-bound forms, respectively, of an AKR (MSMEG_2407) from Mycobacterium smegmatis, a close homologue of the M. tuberculosis enzyme Rv2971, whose function is essential to this bacterium. MSMEG_2407 adopted the triosephosphate isomerase (α/β)₈-barrel fold exhibited by other AKRs. MSMEG_2407 (AKR5H1) bound NADPH via an induced-fit mechanism, in which the NADPH was ligated in an extended fashion. Polar-mediated interactions dominated the interactions with the cofactor, which is atypical of the mode of NADPH binding within the AKR family. Moreover, the nicotinamide ring of NADPH was disordered, and this was attributed to the lack of an “AKR-conserved” bulky residue within the nicotinamide-binding cavity of MSMEG_2407. Enzymatic characterisation of MSMEG_2407 and Rv2971 identified dicarbonyls as a preferred substrate family for hydrolysis, and the frontline antituberculosis drug isoniazid (INH) was shown to inhibit the enzyme activity of both recombinant MSMEG_2407 and Rv2971. However, differences between the affinities of MSMEG_2407 and Rv2971 for dicarbonyls and INH were observed, and this was attributable to amino acid substitutions within the cofactor- and substrate-binding sites. The structures of MSMEG_2407 and the accompanying biochemical characterisation of MSMEG_2407 and Rv2971 provide insight into the structure and function of AKRs from mycobacteria.     

Essential Molecular Determinants for Thyroid Hormone Transport and First Structural Implications for Monocarboxylate Transporter 8

Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. To gain insight into structure-function relations in TH transport, we chose human MCT8 as a paradigm. We systematically performed conventional and liquid chromatography-tandem mass spectrometry-based uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for l-iodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and {"type":"entrez-nucleotide","attrs":{"text":"F21388","term_id":"2060564","term_text":"F21388"}}F21388, potent competitors for TH binding at transthyretin, did not inhibit T₃ transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate interaction. Indeed, alanine mutation of either Arg⁴⁴⁵ (helix 8) or Asp⁴⁹⁸ (helix 10) abrogated T₃ transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows us to rationalize potential interactions of amino acids including those mutated in patients with Allan-Herndon-Dudley syndrome.     

Analyses of the Sequence and Structural Properties Corresponding to Pentapeptide and Large Palindromes in Proteins

The analyses of 3967 representative proteins selected from the Protein Data Bank revealed the presence of 2803 pentapeptide and large palindrome sequences with known secondary structure conformation. These represent 2014 unique palindrome sequences. 60% palindromes are not associated with any regular secondary structure and 28% are in helix conformation, 11% in strand conformation and 1% in the coil conformation. The average solvent accessibility values are in the range between 0–155.28 Ų suggesting that the palindromes in proteins can be either buried, exposed to the solvent or share an intermittent property. The number of residue neighborhood contacts defined by interactions ≤ 3.2 Ǻ is in the range between 0–29 residues. Palindromes of the same length in helix, strand and coil conformation are associated with different amino acid residue preferences at the individual positions. Nearly, 20% palindromes interact with catalytic/active site residues, ligand or metal ions in proteins and may therefore be important for function in the corresponding protein. The average hydrophobicity values for the pentapeptide and large palindromes range between -4.3 to +4.32 and the number of palindromes is almost equally distributed between the negative and positive hydrophobicity values. The palindromes represent 107 different protein families and the hydrolases, transferases, oxidoreductases and lyases contain relatively large number of palindromes.