Structural and Functional Annotation of Hypothetical Protein AVO28_00330 of Yersinia pestis: An In Silico Approach

Yersinia pestis is an infamous gram-negative, coccobacillus enterobacterium responsible for three devastating plague pandemics worldwide. The recent outbreak of this zoonotic disease demands in silico study of the hypothetical proteins for efficient drug and vaccine discovery. As hypothetical proteins constitute a substantial portion of the proteome, it’s essential to annotate them structurally and functionally. The current study characterized physicochemical properties, predicted homology-based 3D structure and annotated functions of the hypothetical protein AVO28_00330 of Y. pestis using a range of bioinformatic tools and softwares. Swiss Model and Phyre2 server were utilized to predict the tertiary model which was minimized energetically using YASARA server. The quality assessment servers found the model as a good one. For future molecular docking analysis, active binding sites were predicted using CASTp. Protein-protein interaction analysis was performed in STRING server. For functional prediction InterPro, Pfam, Motif and other tools were used. The hypothetical protein revealed tricopeptide repeat domain and rubredoxin metal-binding domain which regulates lipopolysaccharide metabolic process in the outer cell membrane which contributes to virulence property of the protein. Therefore, this in silico analysis will improve the current understanding of the protein and aid in the future analysis regarding therapeutic drug and vaccine investigation.

Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum

Background Plasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT. Methods Using the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter’s substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron. Results The combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein. Conclusion The structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

Structural and functional studies of SAV1707 from Staphylococcus aureus elucidate its distinct metal-dependent activity and a crucial residue for catalysis

The metallo-β-lactamase fold is the most abundant metal-binding domain found in two major kingdoms: bacteria and archaea. Despite the rapid growth in genomic information, most of these enzymes, which may play critical roles in cellular metabolism, remain uncharacterized in terms of structure and function. In this study, X-ray crystal structures of SAV1707, a hypothetical metalloenzyme from Staphylococcus aureus, and its complex with cAMP are reported at high resolutions of 2.05 and 1.55 Å, respectively, with a detailed atomic description. Through a functional study, it was verified that SAV1707 has Ni2+-dependent phosphodiesterase activity and Mn2+-dependent endonuclease activity, revealing a different metal selectivity depending on the reaction. In addition, the crystal structure of cAMP-bound SAV1707 shows a unique snapshot of cAMP that reveals the binding mode of the intermediate, and a key residue Phe511 that forms π–π interactions with cAMP was verified as contributing to substrate recognition by functional studies of its mutant. Overall, these findings characterized the relationship between the structure and function of SAV1707 and may provide further understanding of metalloenzymes possessing the metallo-β-lactamase fold.

Characterization of a Novel Variant of the Quinolone-Resistance Gene qnrB (qnrB89) Carried by a Multi-Drug Resistant Citrobacter gillenii Strain Isolated from Farmed Salmon in Chile

The main objective of this study was to characterize using whole-genome sequencing analysis, a new variant of the qnrB gene (qnrB89) carried by a fluoroquinolone-susceptible bacterium isolated from mucus of farmed Salmo salar fingerling in Chile. Citrobacter gillenii FP75 was identified by using biochemical tests and 16S ribosomal gene analysis. Nucleotide and amino acid sequences of the qnrB89 gene exhibited an identity to qnrB of 81.24% and 91.59%, respectively. The genetic environment of qnrB89 was characterized by the upstream location of a sequence encoding for a protein containing a heavy metal-binding domain and a gene encoding for a N-acetylmuramoyl-L-alanine amidase protein, whereas downstream to qnrB89 gene were detected the csp and cspG genes, encoding cold-shock proteins. The qnrB89 gene was located on a large chromosomal contig of the FP75 genome and was not associated with the 10-kb plasmid and class 1 integron harbored by the FP75 strain. This study reports for the first time the carriage of a qnrB gene by the C. gillenii species, and its detection in a bacterial strain isolated from farmed salmon in Chile.

Manipulating the Conformation of 3,2′:6′,3″-Terpyridine in [Cu2(μ-OAc)4(3,2′:6′,3″-tpy)]n 1D-Polymers

We report the preparation and characterization of 4′-([1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (1), 4′-(4′-fluoro-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (2), 4′-(4′-chloro-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (3), 4′-(4′-bromo-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (4), and 4′-(4′-methyl-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (5), and their reactions with copper(II) acetate. Single-crystal structures of the [Cu2(μ-OAc)4L]n 1D-coordination polymers with L = 1–5 have been determined, and powder X-ray diffraction confirms that the single crystal structures are representative of the bulk samples. [Cu2(μ-OAc)4(1)]n and [Cu2(μ-OAc)4(2)]n are isostructural, and zigzag polymer chains are present which engage in π-stacking interactions between [1,1′-biphenyl]pyridine units. 1D-chains nest into one another to give 2D-sheets; replacing the peripheral H in 1 by an F substituent in 2 has no effect on the solid-state structure, indicating that bifurcated contacts (H...H for 1 or H...F for 2) are only secondary packing interactions. Upon going from [Cu2(μ-OAc)4(1)]n and [Cu2(μ-OAc)4(2)]n to [Cu2(μ-OAc)4(3)]n, [Cu2(μ-OAc)4(4)]n, and [Cu2(μ-OAc)4(5)]n·nMeOH, the increased steric demands of the Cl, Br, or Me substituent induces a switch in the conformation of the 3,2′:6′,3″-tpy metal-binding domain, and a concomitant change in dominant packing interactions to py–py and py–biphenyl face-to-face π-stacking. The study underlines how the 3,2′:6′,3″-tpy domain can adapt to different steric demands of substituents through its conformational flexibility.

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

Copper’s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

AscA (YecA) is a molecular chaperone involved in Sec-dependent protein translocation in Escherichia coli

ABSTRACTProteins that are translocated across the cytoplasmic membrane by Sec machinery must be in an unfolded conformation in order to pass through the protein-conducting channel during translocation. Molecular chaperones assist Sec-dependent protein translocation by holding substrate proteins in an unfolded conformation in the cytoplasm until they can be delivered to the membrane-embedded Sec machinery. For example, in Escherichia coli, SecB binds to a subset of unfolded Sec substrates and delivers them to the Sec machinery by interacting with the metal-binding domain (MBD) of SecA, an ATPase required for translocation in bacteria. Here, we describe a novel molecular chaperone involved Sec-dependent protein translocation, which we have named AscA (for accessory Sec component). AscA contains a metal-binding domain (MBD) that is nearly identical to the MBD of SecA. In vitro binding studies indicated that AscA binds to SecB and ribosomes in an MBD-dependent fashion.Saturated transposon mutagenesis and genetics studies suggested that AscA is involved in cell-envelope biogenesis and that its function overlaps with that of SecB. In support of this idea, AscA copurified with a range of proteins and prevented the aggregation of citrate synthase in vitro. Our results suggest that AscA is molecular chaperone and that it enhances Sec-dependent protein translocation by delivering its substrate proteins to SecB.IMPORTANCEThis research describes the discovery of a novel molecular chaperone, AscA (YecA). The function of AscA was previously unknown. However, it contains a small domain, known as the MBD, suggesting it could interact with the bacterial Sec machinery, which is responsible for transporting proteins across the cytoplasmic membrane. The work described this study indicates that the MBD allows AscA to bind to both the protein synthesis machinery and the Sec machinery. The previously function of the previously uncharacterised N-terminal domain is that of a molecular chaperone, which binds to unfolded substrate proteins. We propose that AscA binds to protein substrates as they are still be synthesised by ribosomes in order to channel them into the Sec pathway.

Directing 2D-Coordination Networks: Combined Effects of a Conformationally Flexible 3,2′:6′,3″-Terpyridine and Chain Length Variation in 4′-(4-n-Alkyloxyphenyl) Substituents

The synthesis and characterization of 4′-(4-n-propoxyphenyl)-3,2′:6′,3″-terpyridine is described. Five 2D-coordination networks have been isolated by crystal growth at room temperature from reactions of Co(NCS)2 with 4′-(4-n-alkyloxyphenyl)-3,2′:6′,3″-terpyridines in which the n-alkyl group is ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl in ligands 2–6, respectively. The single-crystal structures of [{Co(2)2(NCS)2}.0.6CHCl3]n, [{Co(3)2(NCS)2}.4CHCl3.0.25H2O]n, [{Co(4)2(NCS)2}.4CHCl3]n, [Co2(5)4(NCS)4]n and [Co(6)2(NCS)2]n have been determined, and powder X-ray diffraction has demonstrated that the single-crystal structures are representative of the bulk materials. Each compound possesses a (4,4) net with Co centres as 4-connecting nodes. For the assemblies containing 2, 3 and 4, the (4,4) net comprises two geometrically different rhombuses, and the nets pack in an ABAB... arrangement with cone-like arrangements of n-alkyloxyphenyl groups being accommodated in a similar unit in an adjacent net. An increase in the n-alkyloxy chain length has two consequences: there is a change in the conformation of the 3,2′:6′,3″-tpy metal-binding domain, and the (4,4) net comprises identical rhombuses. Similarities and differences between the assemblies with ligands 2–6 and the previously reported [{Co(1)2(NCS)2}.3MeOH]n and [{Co(1)2(NCS)2}.2.2CHCl3]n in which 1 is 4′-(4-methoxyphenyl)-3,2′:6′,3″-terpyridine are discussed. The results demonstrate the effects of combining a variable chain length in the 4′-(4-n-alkyloxyphenyl) substituents of 3,2′:6′,3″-tpy and a conformationally flexible 3,2′:6′,3″-tpy metal-binding domain.

Iron is a ligand of SecA-like metal-binding domains in vivo

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

Wilson disease missense mutations in ATP7B affect metal-binding domain structural dynamics

Wilson disease (WD) is caused by mutations in the gene for ATP7B, a copper transport protein that regulates copper levels in cells. A large number of missense mutations have been reported to cause WD but genotype–phenotype correlations are not yet established. Since genetic screening for WD may become reality in the future, it is important to know how individual mutations affect ATP7B function, with the ultimate goal to predict pathophysiology of the disease. To begin to assess mechanisms of dysfunction, we investigated four proposed WD-causing missense mutations in metal-binding domains 5 and 6 of ATP7B. Three of the four variants showed reduced ATP7B copper transport ability in a traditional yeast assay. To probe mutation-induced structural dynamic effects at the atomic level, molecular dynamics simulations (1.5 μs simulation time for each variant) were employed. Upon comparing individual metal-binding domains with and without mutations, we identified distinct differences in structural dynamics via root-mean square fluctuation and secondary structure content analyses. Most mutations introduced distant effects resulting in increased dynamics in the copper-binding loop. Taken together, mutation-induced long-range alterations in structural dynamics provide a rationale for reduced copper transport ability.