Structural analysis of the effects of mutations in Ubl domain of Parkin leading to Parkinson's disease.

Parkinson's disease (PD) is characterized by progressive death of dopamine producing neurons in the substantia nigra pars compacta of the mid brain. Dysfunction of an E3 ligase protein, Parkin, encoded by PARK2 gene, results in accumulation of misfolded proteins in brain cells which lead to the onset of PD. Parkin is a multi-domain protein consisting of N-terminal ubiquitin-like domain (Ubl) followed by RING0, RING1, In Between Ring (IBR) domain and RING2 domain which is present at the C-terminal end of Parkin protein. Ubl domain is the smallest domain of Parkin and is involved in the binding of Parkin with E2 protein molecule required for proper Ubiquitination and functioning of proteins in the brain. Mutations in the Parkin protein are known to be associated with protein dysfunction leading to PD. This study aims to decipher the characteristics and effects of the different mutations in the Ubl domain by an in-silico analysis. The mutations were collected from PDmutDB and COSMIC databases. The pathogenic impacts of amino-acid mutations on Ubl structure and function were analysed by using various computational tools. Due to lack of proper full-chain structure of the Ubl domain, a homology model of the domain was reconstructed using Discovery Studio 2.5 (DS 2.5) software suite. We found that the mutations A31D, A46P, C59F, A46T, E28K, E49K, R42P, R42S, and Q63K were the most deleterious ones which might be associated with the onset of PD. In order to study the dynamic behaviour of the Parkin Ubl domain in cellular environment, molecular dynamics (MD) simulations were carried out using the wild-type and mutant Ubl domains. Our analyses could predict the cellular dynamics of the mutations and therefore might help in predicting the hitherto unknown molecular mechanism of the disease onset and designing precision medicine for the treatment of PD.

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape.

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121-148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121-148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121-148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

Structure based virtual screening of natural products to disrupt the structural integrity of TRAF6 C-terminal domain homotrimer.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ligase which takes part in different cellular pathways. TRAF6 is seen to be highly expressed in various cancers and most importantly is known to drive cancer metastasis. This makes TRAF6 a potential therapeutic target. In our previous studies, we observed that the C-terminal domain of TRAF6 forms a mushroom shaped trimer structure. Lys340 and Glu345 were identified to be the most critical residues in the trimer interface. In this current work, we screened for more than 14000 small molecules derived from various natural sources and they were screened against TRAF6 C-terminal trimer interaction interface to prevent the formation of the interface. All the obtained molecules were tested for their drug-likeliness properties. The ligands which qualified the filter were considered for protein-ligand docking or structure based virtual screening in GOLD 5.2. Pose selection was carried out on the basis of GoldScore and ChemScore function of GOLD 5.2. Top 20 molecules binding the TRAF6 trimeric interface were tested for their ADME properties. From the top 20 molecules, top 3 ligands were chosen based on their abilities to pass the maximum numbers of ADME filters.

Insight into the binding of a synthetic nitro-flavone derivative with human poly (ADP-ribose) polymerase 1.

Flavones are important bioactive compounds, many of which are effective in cancer therapy for their ability to target enzymes related to DNA repair and cell proliferation. In this report, the interaction of a synthetic nitroflavone, 2,4-nitrophenylchromen-4-one (4NCO) with human poly (ADP-ribose) polymerase 1 (hPARP1) was investigated to explore its inhibitory action. Its interaction with hPARP1 was compared with that of other inhibitors through molecular docking studies. Further insight into the 4NCO-hPARP1 interaction was obtained from competitive docking and molecular dynamic simulation studies. In silico mutagenesis studies and per-residue interaction energy calculations were carried out. Quantitative Structure Activity Relationship analysis was also performed to calculate its predictive percent inhibitory activity. Our results indicated that 4NCO exhibited competitive mode of binding to hPARP1. It formed a stable interaction with the protein thereby hindering any further molecular interaction to render it inactive with a predictive inhibition of 96%. It also had good ADMET properties and showed best Autodock binding free energy values compared to other known inhibitors. 4NCO showed good hPARP1 inhibitory properties with higher bioavailability and lower probability of getting effluxed. Development of inhibitors against hPARP1 is important for cell proliferative disorders, where 4NCO can be predicted as a potential new drug.

Glycine rich proline rich protein from Sorghum bicolor serves as an antimicrobial protein implicated in plant defense response.

KEY MESSAGE: Sorghum glycine rich proline rich protein (SbGPRP1) exhibit antimicrobial properties and play a crucial role during biotic stress condition. Several proteins in plants build up the innate immune response system in plants which get triggered during the occurrence of biotic stress. Here we report the functional characterization of a glycine-rich proline-rich protein (SbGPRP1) from Sorghum which was previously demonstrated to be involved in abiotic stresses. Expression studies carried out with SbGPRP1 showed induced expression upon application of phytohormones like salicylic acid which might be the key in fine-tuning the expression level. Upon challenging the Sorghum plants with a compatible pathogen the SbGprp1 transcript was found to be upregulated. SbGPRP1 encodes a 197 amino acid polypeptide which was bacterially-expressed and purified for in vitro assays. Gram-positive bacteria like Bacillus and phytopathogen Rhodococcus fascians showed inhibited growth in the presence of the protein. The NPN assay, electrolytic leakage and SEM analysis showed membrane damage in bacterial cells. Ectopic expression of SbGPRP1 in tobacco plants led to enhanced tolerance towards infection caused by R. fascians. Though the N-terminal part of the protein showed disorderness the C-terminal end was quite capable of forming several α-helices which was correlated with CD spectroscopic analysis. Here, we have tried to determine the structural model for the protein and predicted the association of antimicrobial activity with the C-terminal region of the protein.

Structural Characterization of the Trimerization of TRAF6 Protein Through Molecular Dynamics Simulations.

The tumour necrosis factor (TNF) receptor-associated factor (TRAF) family of proteins having E3 ligase activity are the key molecules involved in cellular immune response pathways. TRAF6 is a unique member of the TRAF superfamily differing from other members of the family, owing to its specific interactions with molecules outside the TNF receptor superfamily. The C-terminal domain of TRAF proteins contains the catalytic residues and are known to be involved in self-oligomerization forming a mushroom-shaped trimeric structure, which is the functional form of the protein. However, the monomeric crystal structure of TRAF6 C-terminal domain has been already determined, but the trimeric structure of the same is still not available. We here applied computational structural modelling and molecular dynamics simulations studies to get insights into the molecular interactions involved in determining the trimeric structure of the TRAF6 C-terminal domain. The non-availability of the trimeric structure of the TRAF6 C-terminal domain prevented the elucidation of the molecular mechanism of many different biological processes. Our results suggest that the trimer complex is transient in nature. The amino acid residues Lys340 and Glu345 in the coiled coil domain in the C-terminus of TRAF6 play a critical role in trimer structure formation. This structural modelling study may therefore be utilized to obtain the experimentally validated trimeric structure of this important protein.

Identification and analyses of natural compounds as potential inhibitors of TRAF6-Basigin interactions in melanoma using structure-based virtual screening and molecular dynamics simulations.

The interaction of the proteins, tumor necrosis factor receptor-associated factor6 (TRAF6) and Basigin (CD147), is known to be associated with the over-expression of matrix metalloproteinases (MMPs) in melanoma cells. MMPs are known to be responsible for melanoma metastasis. Hence, the TRAF6-Basigin complex can act as a potential therapeutic target. In previous studies, amino acid residues Lys340, Lys 384, Glu417 and Glu511 of TRAF6 were identified as the most vital residues on the basis of their contributions to interaction energy, relative solvent accessibility and electrostatic interactions in the TRAF6-Basigin protein-protein interaction (PPI) scheme. In our current work, we performed structure-based virtual screenings of some natural compounds obtained from ZINC database (n = 14509) to search for molecules which can act as inhibitors against the formation of TRAF6-Basigin complex. Three potential inhibitors were identified which were observed to make intermolecular interactions with Lys384 and Glu511 of TRAF6. Molecular dynamics simulation results suggested the substantial pharmacological importance of the ligand molecules as it was observed that there was total destabilization of TRAF6-Basigin complex upon binding of the molecule ZINC02578057. From our studies, we could conclude that the ligands termed as ZINC49048033, ZINC02578057 and ZINC72320240 could have great potentials to act as inhibitors to prevent melanoma metastasis.

Inhibition of TRAF6-Ubc13 interaction in NFkB inflammatory pathway by analyzing the hotspot amino acid residues and protein-protein interactions using molecular docking simulations.

Protein-protein interactions (PPIs) are important in most of the biochemical processes. Hotspot amino acid residues in proteins are the most important contributors for proper protein-protein interactions. Hotspot amino acid residues have been looked down upon as important therapeutic targets in inhibiting PPIs. Interaction between TRAF6 and Ubc13 is a crucial point in the NFkB inflammatory pathway. Dysfunction of the NFkB pathway is associated with numerous human diseases including cancer and neurodenegeration disorders. Ubc13 also interacts specifically to TRAF6 and not with other proteins of the TRAF family and this makes the TRAF6-Ubc13 complex an important target for specific inhibition. Hence, interfering with the TRAF6-Ubc13 association may prove effective in suppressing the NFkB disease pathway. In the present study, we searched the TRAF6-Ubc13 interaction interface to analyze their binding hotspot amino acid residues using various computational techniques. Heterocyclic compounds are known for their medicinal properties. We screened for heterocyclic analogues to the known TRAF6 inhibitor PDTC, to predict a better inhibitor using in silico protein-ligand and protein-protein interaction studies. Our in silico prediction results suggest that tetrahydro-2-thiophenecarbothioamide (Chemspider ID 36027528) binds one of the major hot-spot residues of TRAF6-Ubc13 interface and can be a better alternative in suppressing TRA6-Ubc13 complex formation in chronic inflammation than PDTC.

A structural perspective on the interactions of TRAF6 and Basigin during the onset of melanoma: A molecular dynamics simulation study.

Metastatic melanoma is the most fatal type of skin cancer. The roles of matrix metalloproteinases (MMPs) have well been established in the onset of melanoma. Basigin (BSG) belongs to the immunoglobulin superfamily and is critical for induction of extracellular MMPs during the onset of various cancers including melanoma. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3-ligase that interacts with BSG and mediates its membrane localization, which leads to MMP expression in melanoma cells. This makes TRAF6 a potential therapeutic target in melanoma. We here conducted protein-protein interaction studies on TRAF6 and BSG to get molecular level insights of the reactions. The structure of human BSG was constructed by protein threading. Molecular-docking method was applied to develop the TRAF6-BSG complex. The refined docked complex was further optimized by molecular dynamics simulations. Results from binding free energy, surface properties, and electrostatic interaction analysis indicate that Lys340 and Glu417 of TRAF6 play as the anchor residues in the protein interaction interface. The current study will be helpful in designing specific modulators of TRAF6 to control melanoma metastasis.

A comprehensive computational study on pathogenic mis-sense mutations spanning the RING2 and REP domains of Parkin protein.

Various mutations in PARK2 gene, which encodes the protein parkin, are significantly associated with the onset of autosomal recessive juvenile Parkinson (ARJP) in neuronal cells. Parkin is a multi domain protein, the N-terminal part contains the Ubl and the C-terminal part consists of four zinc coordinating domains, viz., RING0, RING1, in between ring (IBR) and RING2. Disease mutations are spread over all the domains of Parkin, although mutations in some regions may affect the functionality of Parkin more adversely. The mutations in the RING2 domain are seen to abolish the neuroprotective E3 ligase activity of Parkin. In this current work, we carried out detailed in silico analysis to study the extent of pathogenicity of mutations spanning the Parkin RING2 domain and the adjoining REP region by SIFT, Mutation Accessor, PolyPhen2, SNPs and GO, GV/GD and I-mutant. To study the structural and functional implications of these mutations on RING2-REP domain of Parkin, we studied the solvent accessibility (SASA/RSA), hydrophobicity, intra-molecular hydrogen bonding profile and domain analysis by various computational tools. Finally, we analysed the interaction energy profiles of the mutants and compared them to the wild type protein using Discovery studio 2.5. By comparing the various analyses it could be safely concluded that except P437L and A379V mutations, all other mutations were potentially deleterious affecting various structural aspects of RING2 domain architecture. This study is based purely on computational approach which has the potential to identify disease mutations and the information could further be used in treatment of diseases and prognosis.

Comparative Analyses of the Relative Effects of Various Mutations in Major Histocompatibility Complex I-a Way to Predict Protein-Protein Interactions.

Protein-protein interactions (PPIs) play pivotal roles in most of the biological processes. PPI dysfunctions are therefore associated with disease situations. Mutations often lead to PPI dysfunctions, but there are certain other types of mutations which do not cause any appreciable abnormalities. This second type of mutations is called polymorphic mutations. So far, there are many studies that deal with the identification of PPI sites, but clear-cut analyses of the involvements of mutations in PPI dysfunctions are few and far between. We therefore made an attempt to link the appearances of mutations and PPI disruptions. We used major histocompatibility complex as our reference protein complex. We analyzed the mutations leading to the disease amyloidosis and also the other mutations that do not lead to disease conditions. We computed various biophysical parameters like relative solvent accessibility to discriminate between the two different types of mutations. Our analyses for the first time came up with a plausible explanation for the effects of different types of mutations in disease development. Our future plans are to build tools to detect the effects of mutations in disease developments by disrupting the PPIs.

Isolation of pure compound R/J/3 from Pluchea indica (L.) Less. and its anti-amoebic activities against Entamoeba histolytica.

The plant Pluchea indica is known for its anti-inflammatory, anti-ulcer, anti-pyretic, hypoglycemic, diuretic and anti-microbial activities besides many other pharmacological activities. We have isolated and purified seven compounds from the methanolic root extract of this plant by column chromatography. The compounds were identified by spectroscopic analyses. The anti-amoebic activities of the pure compound R/J/3 was investigated against the HM1 strain of Entamoeba histolytica. The compound, R/J/3 showed the most pronounced anti-proliferative activity at a dose of 50 microg/ml. It also showed a marked activity on cell lysis of trophozoites, 4h after administration. The cell lytic activity was compared with metronidazole (5 microg/ml) as positive control.