Molecular dynamics simulation study of Plasmodium falciparum and Escherichia coli SufA: Exploration of conformational changes possibly involved in iron-sulfur cluster transfer.

Iron-sulfur (Fe-S) clusters are one of the earliest known metal complexes in biological molecules. Suf system is one of the Fe-S biogenesis pathways. SufA belongs to the Suf pathway. It is an A-type carrier protein that transfers Fe-S clusters from the scaffold to target proteins. Structural studies were performed for the Suf pathway protein, SufA, in order to explore the conformational changes that probably aid in the transfer of Fe-S clusters to target proteins. Three-dimensional (3D) structure of Plasmodium falciparum (Pf) SufA homodimer was obtained by homology modeling using 3D structure of Escherichia coli (Ec) SufA as template. Molecular dynamics (MD) simulation of Pf SufA and Ec SufA homodimers followed by trajectory and pocket analyses were carried out. A co-ordinated displacement of the homodimeric chains in the interfacial region, resembling a swinging trapeze-like movement was observed. Potential involvement of this swinging trapeze-like movement of the residues belonging to the interfacial region has been proposed as a probable mechanism that assists in the transfer of Fe-S cluster from SufA to apo proteins. This was substantiated by protein-protein interaction studies in Pf SufA by performing molecular docking of 3D conformations of Pf SufA obtained from MD trajectory at every 1 ns interval with Pf ferredoxin.Communicated by Ramaswamy H. Sarma.

Molecular dynamics of the membrane interaction and localisation of prodigiosin.

The tripyrrolic antibiotic prodigiosin causes diverse reactions on its targets like energy spilling, membrane leakage, loss of motility and phototoxicity. It has bacteriostatic, bactericidal, anti-fungal, anti-cancer and immunosuppressive properties. Most of the functions suggest the role of prodigiosin in membrane disruption but the exact mechanism remains unknown. A molecular dynamics study was performed to understand the interactions of prodigiosin with the membrane. It was seen that prodigiosin from the solvent enters the membrane immediately either individually or as small clusters. Prodigiosin clusters with more than eight molecules do not appear to enter the membrane. Upon entry, the molecules orient themselves along the membrane-water interface with the pyrrole rings interacting with lipid head groups and with water. This orientation is stabilised by hydrogen bonding and hydrophobic interactions. The presence of prodigiosin molecules in the membrane changes the local lipid architecture and reduces the solvent accessibility of the membrane. The membrane fluidity, thickness or area per lipid head are largely unaffected. This suggests that prodigiosin could cause most damage in the vicinity of a membrane protein and thus could also explain the reason for varied effects on the targets.

Thalidomide and Its Analogs Differentially Target Fibroblast Growth Factor Receptors: Thalidomide Suppresses FGFR Gene Expression while Pomalidomide Dampens FGFR2 Activity.

Thalidomide is an infamous teratogen and it is continuously being explored for its anticancer properties. Fibroblast growth factor receptors (FGFRs) are implicated in embryo development and cancer pathophysiology. With striking similarities observed between FGFR implicated conditions and thalidomide embryopathy, we hypothesized thalidomide targets FGFRs. We utilized three different cell lines and chicken embryo model to investigate the effects of thalidomide and analogs on FGFR expression. We performed molecular docking, KINOMEscan analysis, and kinase activity assays to study the drug-protein interactions. The expression of FGFR1 and FGFR2 was differentially regulated by all the three drugs in cells as well as in developing organs. Transcriptome analysis of thalidomide-treated chick embryo strongly suggests the modulation of FGFR signaling and key transcription factors. Corroboration with previous studies suggests that thalidomide might affect FGFR expression through the transcription factor, E2F1. At the protein level, molecular docking predicted all three analogs to interact with lysine residue at 517th and 508th positions of FGFR2 and FGFR3, respectively. This lysine coordinates the ATP binding site of FGFR, thus hinting at the possible perturbation of FGFR activity by thalidomide. Kinome analysis revealed that kinase activities of FGFR2 and FGFR3 (G697C) reduced by 31% and 65%, respectively, in the presence of 10 µM thalidomide. Further, we checked and confirmed that the analogs inhibited the FGFR2 kinase activity in a dose-dependent manner. This study suggests that FGFRs could be potential targets of thalidomide and the two analogs, and also endorses the link between the teratogenicity and antitumor activities of the drugs.

Comparative pan genome analysis of oral Prevotella species implicated in periodontitis.

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a "Por_secre_tail" domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.

Molecular dynamics simulations of lectin domain of FimH and immunoinformatics for the design of potential vaccine candidates.

Adhesion of uropathogenic E. coli (UPEC) to uroepithelial cell receptors is facilitated through the lectin domain of FimH adhesin. In the current study, Molecular Dynamics (MD) simulations were performed for the lectin domain of FimH from UPEC J96. The high affinity state lectin domain was found to be stable and rigid during the simulations. Further, based on conserved subsequences around one of the disulfide forming cysteines, two sequence motifs were designed. An immunoinformatics approach was utilized to identify linear and discontinuous epitopes for the lectin domain of FimH. We propose that the accessibility of predicted epitopes should also be assessed in a dynamic aqueous environment to evaluate the potential of vaccine candidates. Since MD simulation data enables assessing the accessibility in a dynamic environment, we evaluated the accessibility of the top ranked discontinuous and linear epitopes using structures obtained at every nanosecond (ns) in the 1-20 ns MD simulation timeframe. Knowledge gained in this study has a potential utility in the design of vaccine candidates for Urinary Tract Infection (UTI).

Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors.

Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin.

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase.

BACKGROUND AND PURPOSE: Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets. EXPERIMENTAL APPROACH: Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC). KEY RESULTS: Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis. CONCLUSION AND IMPLICATIONS: Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.

Independent origin of 185delAG BRCA1 mutation in an Indian family.

BRCA1 and BRCA2 are the two major breast cancer susceptibility genes. We tested members of a family with multiple cases of breast cancer, for mutations in the BRCA1 gene. Analysis of the PCR amplicons of all the exons (22) of the BRCA1 gene using conformation sensitive gel electrophoresis (CSGE) revealed a heteroduplex band pattern in exon 2 of the proband (III-3) in this family. The amplicon was further sequenced to assess the nature of the mutation, which revealed a deletion of AG nucleotides at the 185th position (185delAG). The two base pair deletion introduces a stop codon at the 39th amino acid residue. A similar analysis was carried out on other extended family members to evaluate their allelic status. We detected the same deletion in 7 of the 19 members tested. Two of them are males. Haplotype analysis suggested an independent origin for this mutation. Our study highlights the importance of testing hereditary cases of breast/ovarian cancer for BRCA1 mutations in extended families in order to identify high-risk individuals at a pre-clinical stage and provide genetic counseling.

Understanding mutations and protein stability through tripeptides.

A novel methodology to predict the local conformational changes in a protein as a consequence of missense mutations is proposed. A pentapeptide at the locus of mutation plays the dominant role and it is analyzed in terms of tripeptides. A measure for spatial and temporal fluctuations in a pentapeptide is devised and validated. The method does not involve any prior knowledge of structural templates from sequence homology studies. Structural deformations can be predicted with about 70-80% reliability in any protein. Disease causing mutations and benign mutations have been addressed. In particular, p53, retinoblastoma protein and lipoprotein lipase are studied in detail.

Functionally specified protein signatures distinctive for each of the different blue copper proteins.

BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section. CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences.

Mutation analysis of congenital cataracts in Indian families: identification of SNPS and a new causative allele in CRYBB2 gene.

PURPOSE: To study some functional candidate genes in cataract families of Indian descent. METHODS: Nine Indian families, clinically documented to have congenital/childhood cataracts, were screened for mutations in candidate genes such as CRYG (A-->D), CRYBB2, and GJA8 by PCR analyses and sequencing. Genomic DNA samples of either probands or any representative affected member of each family were PCR amplified and sequenced commercially. Documentation of single nucleotide polymorphisms (SNPs) and candidate mutations was done through BLAST SEARCH (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?). RESULTS: Several single nucleotide polymorphisms in CRYG, CRYBB2, and GJA8 genes were observed. Because they do not co-segregate with the phenotype, they were excluded as candidates for the cataract formation in these patients. However, a substitution (W151C in exon 6 of CRYBB2) was identified as the most likely causative mutation underlying the phenotype of central nuclear cataract in all affected members of family C176. Protein structural interpretations demonstrated that no major structural alterations could be predicted and that even the hydrogen bonds to the neighboring Leu166 were unchanged. Surprisingly, hydropathy analysis of the mutant betaB2-crystallin featuring the amino acids at position 147 to 155, further increased the hydrophobicity, which might impair the solubility of the mutant protein. Finally, the Cys residue at position 151 might possibly be involved in intramolecular disulphide bridges with other cysteines during translation, possibly leading to dramatic structural changes. CONCLUSIONS: Exon 6 of CRYBB2 appears to be a critical region susceptible for mutations leading to lens opacity.