Integron Gene Cassettes: A Repository of Novel Protein Folds with Distinct Interaction Sites

Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites), as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.13 page(s

Human protein reference database—2006 update

Human Protein Reference Database (HPRD) () was developed to serve as a comprehensive collection of protein features, post-translational modifications (PTMs) and protein–protein interactions. Since the original report, this database has increased to >20 000 proteins entries and has become the largest database for literature-derived protein–protein interactions (>30 000) and PTMs (>8000) for human proteins. We have also introduced several new features in HPRD including: (i) protein isoforms, (ii) enhanced search options, (iii) linking of pathway annotations and (iv) integration of a novel browser, GenProt Viewer (), developed by us that allows integration of genomic and proteomic information. With the continued support and active participation by the biomedical community, we expect HPRD to become a unique source of curated information for the human proteome and spur biomedical discoveries based on integration of genomic, transcriptomic and proteomic data

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund

I ‘Masque’ nuziali di Thomas Campion: tra rituali galanti e kermesse

Sul contributo interdisciplinare dato da Thomas Campion, poeta, compositore e autore di Masque, all'evoluzione in ambito giacomiano di quest'ultimo genere di spettacolo teatrale

Large scale expression and purification of secreted mouse hephaestin

Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system

Structural and functional analysis of a FeoB A143S G5 loop mutant explains the accelerated GDP release rate

GTPases (Gproteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell division, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric G proteins, the interaction with a membrane-bound Gprotein-coupled receptor catalyzes the release of GDP from the G subunit. Structural and functional studies have implicated one of the nucleotide binding sequence motifs, the G5 motif, as playing an integral part in this release mechanism. Indeed, a G(s) G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a Gprotein-coupled receptor catalyzed release state. In the present study, we investigate the role of the equivalent residue in the G5 motif (residue A143) in the prokaryotic membrane protein FeoB from Streptococcus thermophilus, which includes an N-terminal soluble G protein domain. The structure of this domain has previously been determined in the apo and GDP-bound states and in the presence of a transition state analogue, revealing conformational changes in the G5 motif. The A143 residue was mutated to a serine and analyzed with respect to changes in GTPase activity, nucleotide release rate, GDP affinity and structural alterations. We conclude that the identity of the residue at this position in the G5 loop plays a key role in the nucleotide release rate by allowing the correct positioning and hydrogen bonding of the nucleotide base

Oxidase activity of mouse HEPH.

<p>In all assays WT mouse serum and serum from a mouse CpKO were used as control. Details can be found in the main text. <b>A</b>, An oxidase assay monitoring the oxidation of <i>p</i>PD (increase of A₅₃₀). The rate of HEPH catalysed oxidation reaction increased with an increasing protein concentration. <b>B</b>, <i>p</i>PD oxidation by HEPH was inhibited by the copper-specific chelator D-penicillamine (D-P). <b>C</b>, A ferrozine-based assay was also used to follow the oxidation of Fe²⁺ to Fe³⁺ by HEPH (monitored as a decrease of A₅₇₀). Similarly to the <i>p</i>PD assay, an increased catalytic activity can be observed with an increasing amount of HEPH present. <b>D</b>, The catalytic activity was, as in the pPD assay, inhibited by addition of D-P. <b>E</b>, A velocity versus substrate concentration curve used to obtain <i>K</i>_m and <i>V</i>_max for HEPH. Error bars represents 1 S.D, N = 3.</p

Hephaestin architecture and construct design.

<p><b>A</b>, Schematic illustration of HEPH function. Ferroportin (FPN) exports divalent iron into plasma, where HEPH catalyses the conversion to trivalent metal. <b>B</b>, The structure of ceruloplasmin shown in cartoon representation, with the three copper binding domains coloured in different shades of grey. Copper ions are shown as orange spheres. In ceruloplasmin, 3 peripheral Type I copper sites shuttle electrons from the Fe²⁺—Fe³⁺ oxidation, with the electrons being transferred to the trinuclear copper cluster. <b>C</b>, The overall construct design used in this study.</p