Aromatic Interactions Drive the Coupled Folding and Binding of the Intrinsically Disordered Sesbania mosaic Virus VPg Protein.

The plant Sesbania mosaic virus [a (+)-ssRNA sobemovirus] VPg protein is intrinsically disordered in solution. For the virus life cycle, the VPg protein is essential for replication and for polyprotein processing that is carried out by a virus-encoded protease. The nuclear magnetic resonance (NMR)-derived tertiary structure of the protease-bound VPg shows it to have a novel tertiary structure with an α-ß-ß-ß topology. The quaternary structure of the high-affinity protease-VPg complex (≈27 kDa) has been determined using HADDOCK protocols with NMR (residual dipolar coupling, dihedral angle, and nuclear Overhauser enhancement) restraints and mutagenesis data as inputs. The geometry of the complex is in excellent agreement with long-range orientational restraints such as residual dipolar couplings and ring-current shifts. A "vein" of aromatic residues on the protease surface is pivotal for the folding of VPg via intermolecular edge-to-face π···π stacking between Trp271 and Trp368 of the protease and VPg, respectively, and for the CH···π interactions between Leu361 of VPg and Trp271 of the protease. The structure of the protease-VPg complex provides a molecular framework for predicting sites of important posttranslational modifications such as RNA linkage and phosphorylation and a better understanding of the coupled folding upon binding of intrinsically disordered proteins. The structural data presented here augment the limited structural data available on viral proteins, given their propensity for structural disorder.

Development of pepper vein banding virus chimeric virus-like particles for potential diagnostic and therapeutic applications.

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

EpsN from Bacillus subtilis 168 has UDP-2,6-dideoxy 2-acetamido 4-keto glucose aminotransferase activity in vitro.

The gene epsN of Bacillus subtilis 168 was cloned and overexpressed in Escherichia coli. Purified recombinant EpsN is shown to be a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase by absorption spectroscopy, l-cycloserine inhibition and reverse phase HPLC studies. EpsN catalyzes the conversion of UDP-2,6-dideoxy 2-acetamido 4-keto glucose to UDP-2,6-dideoxy 2-acetamido 4-amino glucose. Lys190 was found by sequence comparison and site-directed mutagenesis to form Schiff base with PLP. Mutagenesis studies showed that, in addition to Lys190, Ser185, Glu164, Gly58 and Thr59 are essential for aminotransferase activity.

EpsM from Bacillus subtilis 168 has UDP-2,4,6-trideoxy-2-acetamido-4-amino glucose acetyltransferase activity in vitro.

Bacillus subtilis 168 EpsM (UniProt id P71063) has been electronically annotated as putative acetyltransferase in the UniProt database. The gene epsM was cloned and overexpressed in E. coli with an N-terminal GST tag. The purified fusion protein was shown by absorption spectroscopy, autoradiography and reverse phase HPLC to catalyse the conversion of UDP-2,4,6-trideoxy-2-acetamido-4-amino glucose to UDP-2,4,6-trideoxy-2,4-diacetamido glucose, commonly known as N,N'-diacetylbacillosamine, using acetyl coenzyme A as the donor substrate. His146 was shown by site-directed mutagenesis to be essential for acetyltransferase activity. It is hypothesized that EpsC (NAD+ dependent UDP GlcNAc 4,6-dehydratase), EpsN (PLP dependent aminotransferase) and EpsM, all of which are part of the eps operon, are involved in the biosynthesis of N,N'-diacetylbacillosamine.

In vitro characterization of N-terminal truncated EpsC from Bacillus subtilis 168, a UDP-N-acetylglucosamine 4,6-dehydratase.

Bacillus subtilis 168 EpsC is annotated as "Probable polysaccharide biosynthesis protein" in the SwissProt database. epsC is part of the eps operon, thought to be involved in the biosynthesis of exopolymeric substances (EPS). The present study was undertaken to determine the molecular function of EpsC. Sequence analysis of EpsC suggested the presence of a transmembrane domain. Two N-terminal deletion mutants in which residues 1-89 (EpsC89) and 1-115 (EpsC115) are deleted were cloned and overexpressed. Enzyme activity and substrate preferences were investigated by reverse phase HPLC, surface plasmon resonance (SPR) spectroscopy and absorption spectroscopy. These data show that EpsC has UDP-GlcNAc 4,6-dehydratase activity in vitro. Purified recombinant proteins were found to utilise UDP-Glc and TDP-Glc also as substrates. In addition, EpsC115 could utilise UDP-Gal and UDP-GalNAc as substrates whereas EpsC89 could only bind these two sugar nucleotides. These results show that deletion of a longer N-terminal region broadens substrate specificity. These broadened specificity is perhaps an outcome of the deletion of the putative transmembrane domain and may not be present in vivo. EpsC, together with the aminotransferase EpsN (Kaundinya CR et al., Glycobiology, 2018) and acetyltransferase EpsM (unpublished data), appears to be involved in the biosynthesis of N,N'-diacetylbacillosamine.

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles.

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn(2+) ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

Structural similarities between SAM and ATP recognition motifs and detection of ATP binding in a SAM binding DNA methyltransferase.

S-adenosylmethionine (SAM) is a ubiquitous co-factor that serves as a donor for methylation reactions and additionally serves as a donor of other functional groups such as amino and ribosyl moieties in a variety of other biochemical reactions. Such versatility in function is enabled by the ability of SAM to be recognized by a wide variety of protein molecules that vary in their sequences and structural folds. To understand what gives rise to specific SAM binding in diverse proteins, we set out to study if there are any structural patterns at their binding sites. A comprehensive analysis of structures of the binding sites of SAM by all-pair comparison and clustering, indicated the presence of 4 different site-types, only one among them being well studied. For each site-type we decipher the common minimum principle involved in SAM recognition by diverse proteins and derive structural motifs that are characteristic of SAM binding. The presence of the structural motifs with precise three-dimensional arrangement of amino acids in SAM sites that appear to have evolved independently, indicates that these are winning arrangements of residues to bring about SAM recognition. Further, we find high similarity between one of the SAM site types and a well known ATP binding site type. We demonstrate using in vitro experiments that a known SAM binding protein, HpyAII.M1, a type 2 methyltransferase can bind and hydrolyse ATP. We find common structural motifs that explain this, further supported through site-directed mutagenesis. Observation of similar motifs for binding two of the most ubiquitous ligands in multiple protein families with diverse sequences and structural folds presents compelling evidence at the molecular level in favour of convergent evolution.

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus.

Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface.

BACKGROUND: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. RESULTS: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K(m) in the order of acetate > propionate > formate. Further, the Km for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg(2+) could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn(2+) and Co(2+)), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic ßßßαßαßα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes. CONCLUSIONS: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

Functional Characterization of Pepper Vein Banding Virus-Encoded Proteins and Their Interactions: Implications in Potyvirus Infection.

Pepper vein banding virus (PVBV) is a distinct species in the Potyvirus genus which infects economically important plants in several parts of India. Like other potyviruses, PVBV encodes multifunctional proteins, with several interaction partners, having implications at different stages of the potyviral infection. In this review, we summarize the functional characterization of different PVBV-encoded proteins with an emphasis on their interaction partners governing the multifunctionality of potyviral proteins. Intrinsically disordered domains/regions of these proteins play an important role in their interactions with other proteins. Deciphering the function of PVBV-encoded proteins and their interactions with cognitive partners will help in understanding the putative mechanisms by which the potyviral proteins are regulated at different stages of the viral life-cycle. This review also discusses PVBV virus-like particles (VLPs) and their potential applications in nanotechnology. Further, virus-like nanoparticle-cell interactions and intracellular fate of PVBV VLPs are also discussed.

Corrigendum: Biological Significance of Marine Actinobacteria of East Coast of Andhra Pradesh, India.

[This corrects the article DOI: 10.3389/fmicb.2017.01201.].

Intracellular trafficking and endocytic uptake pathway of Pepper vein banding virus-like particles in epithelial cells.

Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.

Structural and functional studies on Salmonella typhimurium pyridoxal kinase: the first structural evidence for the formation of Schiff base with the substrate.

A large number of enzymes depend on the ubiquitous cofactor pyridoxal 5' phosphate (PLP) for their activity. Pyridoxal kinase (PLK) is the key enzyme involved in the synthesis of PLP from the three forms of vitamin B6 via the salvage pathway. In the present work, we determined the unliganded structure of StPLK in a monoclinic form and its ternary complex with bound pyridoxal (PL), ADP and Mg2+ in two different tetragonal crystal forms (Form I and Form II). We found that, in the ternary complex structure of StPLK, the active site Lys233 forms a Schiff base linkage with the substrate (PL). Although formation of a Schiff base with the active site Lys229 was demonstrated in the Escherichia coli enzyme based on biochemical studies, the ternary complex of StPLK represents the first crystal structure where the Schiff bond formation has been observed. We also identified an additional site for PLP binding away from the active site in one of the ternary complexes (crystal Form I), suggesting a probable route for the product release. This is the first ternary complex structure where the modeled γ-phosphate of ATP is close enough to PL for the phosphorylation of the substrate. StPLK prefers PL over pyridoxamine as its substrate and follows a sequential mechanism of catalysis. Surface plasmon resonance studies suggest that StPLK interacts with apo-PLP-dependent enzymes with µm affinity supporting the earlier proposed direct transfer mechanism of PLP from PLK to PLP-dependent enzymes.

Mapping the domain of interaction of PVBV VPg with NIa-Pro: Role of N-terminal disordered region of VPg in the modulation of structure and function.

VPg-Pro is involved in polyprotein processing, therefore its regulation is important for a successful potyviral infection. We report here that the N-terminal disordered region of VPg forms the domain of interaction with NIa-Pro. This region is also demonstrated to be responsible for modulating the protease activity of VPg-Pro, both in cis and trans. The disordered nature of VPg is elicited by the N-terminal 22 residues as removal of these residues (∆N22 VPg) brought about gross structural and conformational changes in the protein. Interestingly, ∆N22 VPg gained ATPase activity which suggested the presence of autoinhibitory motif within the N-terminal region of VPg. The autoinhibition gets relieved upon interaction of VPg with NIa-Pro or removal of the inhibitory motif. Thus, the N-terminal 22 residues of VPg qualify as molecular recognition feature (MoRF), regulating both protease and ATPase activity of VPg-Pro as well as forming the domain of interaction with other viral/host proteins.

Biological Significance of Marine Actinobacteria of East Coast of Andhra Pradesh, India.

An attempt was made to identify actinobacterial strains present in the marine soil of East Coast regions viz., Chirala, Bapatla, and Peddaganjam, Andhra Pradesh; Kanyakumari, Tamil Nadu and Goa, Goa along with the study of their antimicrobial potential. Eight out of 73 actinobacterial strains isolated from these regions showed strong antimicrobial activity against Gram positive bacteria, Gram negative bacteria, and Candida albicans. Molecular identification (16S rRNA analysis) of the eight strains revealed that they belong to Dietzia sp., Kocuria sp., Nocardiopsis sp., and Streptomyces spp. ISP (International Streptomyces project) -1, ISP-2 and starch casein media supported high antimicrobial potential after 5-6 days of growth. Production of antimicrobials by the strains varied significantly with different carbon and nitrogen sources. Gas chromatography mass spectrometry (GCMS) analysis of volatile compounds produced by the strains illustrated an array of antimicrobial compounds such as 1, 2-benzene dicarboxylic acid, 2-piperidinone, pyrrolo[1,2-a]pyrazine-1,4-dion, phenyl ethyl alcohol, 3-phenyl propionic acid etc. Ours is the first report on the study and detection of above mentioned antimicrobial metabolites from Dietzia sp. (A3), Kocuria sp. (A5), and Nocardiopsis sp. (A7). By sequence based analysis for secondary metabolites, non-ribosomal peptide synthetase (NRPS) gene cluster was noticed in six strains (A2, A3, A4, A6, A7, and A8) and none of them had polyketide synthase (PKS) system. The present study intimates the biological potentiality of the actinobacterial strains isolated from East Coast of Andhra Pradesh, India which reveals further scope to investigate new bioactive compounds from them by employing both natural product chemistry and modern biotechnological aspects.

A novel viral RNA helicase with an independent translation enhancement activity.

RNA helicases have not been identified among negative sense RNA viruses. In this study, it is shown that Nonstructural protein (NSs) of Groundnut bud necrosis virus (GBNV) acts as a Mg(2+) - and ATP-dependent bipolar RNA helicase. Biophysical and biochemical analysis of the deletion mutants (NΔ124 NSs, CΔ80 NSs) revealed that both the N- and C-terminal residues are required for substrate binding, oligomerization and helicase activity, but are dispensable for ATPase activity. Interestingly, NSs could enhance the translation of RNA (~ 10-fold) independent of its helicase activity. This is the first report of a RNA helicase from negative strand RNA viruses.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the ßH-ßI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication.

Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp-p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, CΔ43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp.