Structural insight into DFMO resistant ornithine decarboxylase from Entamoeba histolytica: an inkling to adaptive evolution

Background: Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO. Methodology/Principal Finding: The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure. Conclusions/Significance: We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI

Biochemical, mutational and in silico structural evidence for a functional dimeric Form of the ornithine decarboxylase from entamoeba histolytica

Background Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state. Methodology and Results In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ~0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer. Conclusion To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption

Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme

ABSTRACT: Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-A resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls

Structural and functional studies of alphavirus replication proteins, nsP1 and nsP4

Alphaviruses encode four replication proteins (nsP1-nsP4) that are an integral part of the viral RNA replication complex. nsP4 is the RNA-dependent RNA polymerase (RdRp) that is essential for viral RNA synthesis. To pursue structural and functional studies of the RdRp, Sindbis virus (SINV) nsP4 was produced in E. coli. Due to degradation problems, expression of the full-length protein was unsuccessful. However, the core catalytic domain of nsP4 (Δ97nsP4, a deletion of the N-terminal 97 amino acids) was stably expressed and purified to homogeneity. We have identified that purified Δ97nsP4 possesses terminal adenylyl transferase (TATase) activity, because it catalyzed the specific addition of adenine to the 3\u27 end of acceptor RNA in the presence of divalent cations. Furthermore, Δ97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP and dATP) to the acceptor RNA in the absence or presence of all other nucleotides. Δ97nsP4 possessing a GDD to GAA mutation inactivates the TATase activity. However, a GDD to SNN mutation did not inactivate the enzyme but reduced its activity to ∼45% of wild-type in the presence of Mg+2. Furthermore, the GDD to SNN mutation had no effect on the TATase activity in the presence of Mn+2 ions. Identification of Δ97nsP4 TATase activity suggests a novel function of nsP4 in polyadenylation of the alphavirus genome. A high throughput experiment was also performed for cloning and expression of nsP4 from various alphaviruses. The core catalytic domain of nsP4 from Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV) were expressed and purified successfully. nsP1, another alphavirus replication protein, is the capping enzyme containing S-adenosyl-L-methionine-dependent methyltransferase (MTase) and divalent cation-dependent guanylyltransferase (GTase) activities. SINV nsP1 was expressed in E. coli and purified. We demonstrated that the recombinant SINV nsP1 was enzymatically active by performing nsP1 MTase and GTase assays. Purified replication proteins (nsP1 and nsP4) were used for crystallization trials, but crystals were not observed perhaps due to the conformational heterogeneity of the samples. However, some needle-like crystals were produced from SINV nsP4 (98-599 residues) and further optimization of the crystallization condition may produce good quality protein crystals for structural studies

Prospects for mucosal vaccine: shutting the door on SARS-CoV-2

The sudden emergence of a highly transmissible and pathogenic coronavirus SARS-CoV-2 in December 2019 from China and its rapid global spread has posed an international health emergency. The rapid development of an effective vaccine is imperative to control the spread of SARS-CoV-2. A number of concurrent efforts to find an effective therapeutic agent or vaccine for COVID-19 (coronavirus disease 2019) are being undertaken globally. Oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. As evidenced by studies on similar coronaviruses (SARS-CoV and MERS-CoV), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against SARS-CoV-2. This article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic

Identification of SARS-CoV-2 Cell Entry Inhibitors by Drug Repurposing Using in Silico Structure-Based Virtual Screening Approach

The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) associated Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to angiotensin converting enzyme 2 (ACE2). The virus specific molecular interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure towards exponentially expending COVID-19. Thereto, high-throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the S-protein and ACE2 host cell receptor. Primary screening identified a few promising drugs for both the targets, which were further analyzed in details by their binding energy, binding modes through molecular docking, dynamics and simulations. Evidently, Eptifibatide acetate, TNP, GNF5, GR 127935 hydrochloride hydrate and RS504393 were found binding to virus binding motifs of ACE2 receptor. Additionally, KT185, KT203 GSK1838705A, BMS195614, and RS504393 were identified to bind at the receptor binding site on the viral S-protein. These identified drug molecules may effectively assist in controlling the rapid spread of SARS-COV-2 by not only potentially inhibiting the virus at entry step but also as anti-inflammatory agents which could impart relief in lung injuries. Timely identification and determination of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-COV-2 inhibition by these drugs could save lives is justified

Computational Guided Identification of Novel Potent Inhibitors of NTD-N-Protein of SARS-CoV-2

The Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 virus has raised severe health problems in china and across the world as well. CoVs encode the nucleocapsid protein (N-protein), an essential RNA-binding protein that performs different roles throughout the virus replication cycle and forms the ribonucleoprotein complex with viral RNA using the N-terminal domain (NTD) of N-protein. Recent studies have shown that NTD-N-protein is a legitimate target for the development of antiviral drugs against human CoVs. Owing to the importance of NTD, the present study focuses on targeting the NTD-N-protein from SARS-CoV-2 to identify the potential compounds. The pharmacophore model has been developed based on the guanosine monophosphate (GMP), a RNA substrate and further pharmacophore-based virtual screening was performed against ZINC database. The screened compounds were filtered by analysing the in silico ADMET properties and drug-like properties. The pharmacokinetically screened compounds (ZINC000257324845, ZINC000005169973, and ZINC000009913056) were further scrutinized through computational approaches including molecular docking and molecular dynamics simulations and revealed that these compounds exhibited good binding affinity as compared to GMP and provide stability to their respective complex with the NTD. Our findings could disrupt the binding of viral RNA to NTD, which may inhibit the essential functions of NTD. These findings may further provide an impetus to develop the novel and potential inhibitor against SARS-CoV-2.</p

Crystal Structure of Aura Virus Capsid Protease and Its Complex with Dioxane: New Insights into Capsid-Glycoprotein Molecular Contacts.

The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphavirus life cycle that is essential to virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimics the hydrophobic pyrollidine ring of Pro405 in the loop region of E2

Cellular uptake of metal oxide-based nanocomposites and targeting of chikungunya virus replication protein nsP3

BACKGROUND: Emergence of new pathogenic viruses along with adaptive potential of RNA viruses has become a major public health concern. Hence it becomes even more important to explore and evaluate the antiviral properties of nanocomposites which is an ever-evolving field of medical biology. METHODS: In this study, series of metal/metal oxide (Ag/NiO : NiO, AN-5%, AN-10% and AN-15%) and ternary metal oxide nanocomposites (Ag2O/NiO/ZnO : N/Z, A/N/Z-1, A/N/Z-2 and A/N/Z-3) have been synthesized and characterized. Cellular uptake of nanocomposites was confirmed by ICP-MS. RESULTS: Intriguingly, molecular docking of metal oxides in the active site of nsP3 validated the binding of nanocomposites to chikungunya virus replication protein nsP3. In-vitro antiviral potential of nanocomposites were tested by performing plaque reduction assay, cytopathic effect (CPE) analysis and qRT-PCR. The nanocomposites showed significant reduction in virus titre. Half-maximal inhibitory concentration (IC50) for A/N/Z-3 and AN-5% were determined to be 2.828 and 3.277 g/mL, respectively. CPE observation and qRT-PCR results were consistent with the data obtained from plaque reduction assay for A/N/Z-3 and AN-5%. CONCLUSION: These results, have opened new avenues for development of nanocomposites based antiviral therapies