Computational investigation on the conformational dynamics of C-terminal truncated α-synuclein bound to membrane.

Accelerated progression rates in Parkinson's disease (PD) have been linked to C-terminal domain (CTD) truncations of monomeric α-Synuclein (α-Syn), which have been suggested to increase amyloid aggregation in vivo and in vitro. In the brain of PD patients, CTD truncated α-Syn was found to have lower cell viability and tends to increase in the formation of fibrils. The CTD of α-Syn acts as a guard for regulating the normal functioning of α-Syn. The absence of the CTD may allow the N-terminal of α-Syn to interact with the membrane thereby affecting the normal functioning of α-Syn, and all of which will affect the etiology of PD. In this study, the conformational dynamics of CTD truncated α-Syn (1-99 and 1-108) monomers and their effect on the protein-membrane interactions were demonstrated using the all-atom molecular dynamics (MD) simulation method. From the MD analyses, it was noticed that among the two truncated monomers, α-Syn (1-108) was found to be more stable, shows rigidness at the N-terminal region and contains a significant number of intermolecular hydrogen bonds between the non-amyloid ß-component (NAC) region and membrane, and lesser number of extended strands. Further, the bending angle in the N-terminal domain was found to be lesser in the α-Syn (1-108) in comparison with the α-Syn (1-99). Our findings suggest that the truncation on the CTD of α-Syn affects its interaction with the membrane and subsequently has an impact on the aggregation.Communicated by Ramaswamy H. Sarma.

Prediction of interface between regions of varying degrees of order or disorderness in intrinsically disordered proteins from dihedral angles.

Intrinsically disordered proteins (IDPs) are proteins that do not form uniquely defined three-dimensional (3-D) structures. Experimental research on IDPs is difficult since they go against the traditional protein structure-function paradigm. Although there are several predictors of disorder based on amino acid sequences, but very limited based on the 3-D structures of proteins. Dihedral angles have a significant role in predicting protein structure because they establish a protein's backbone, which, coupled with its side chain, establishes its overall shape. Here, we have carried out atomistic Molecular Dynamics (MD) simulations on four different proteins: one ordered protein (Monellin), two partially disordered proteins (p53-TAD and Amyloid beta (Aß1-42) peptide), and one completely disordered protein (Histatin 5). The MD simulation trajectories for the corresponding four proteins were used to conduct dihedral angle (ϕ and ѱ) analysis. Then, the average dihedral angles for each of the residues were calculated and plotted against the residue index. We noticed steep rises or falls in the average ϕ value at certain locations in the plot. These sudden shifts in the average ϕ value reflect the interface between regions of varying degrees of order or disorderness in intrinsically disordered proteins. Using this method, the probable conformer of a protein with a higher degree of disorder can be found among the ensembles of structures sampled during the MD simulations. The results of our study offer new understandings on precisely identifying regions of various degrees of disorder in intrinsically disordered proteins.Communicated by Ramaswamy H. Sarma.

Computational investigation on the effect of the peptidomimetic inhibitors (NPT100-18A and NPT200-11) on the α-synuclein and lipid membrane interactions.

Parkinson's disease (PD) is associated with α-synuclein (α-Syn), a presynaptic protein that binds to cell membranes. The molecular pathophysiology of PD most likely begins with the binding of α-Syn to membranes. Recently, two peptidomimetic inhibitors (NPT100-18A and NPT200-11) were identified to potentially interact with α-Syn and affect the interaction of α-Syn with the membrane. In this study, the effect of the two peptidomimetic inhibitors on the α-Syn-membrane interaction was demonstrated. DFT calculations were performed for optimization of the two inhibitors, and the nucleophilicity (N) and electrophilicity (ω) of NPT100-18A and NPT200-11 were calculated to be 3.90 and 3.86 (N); 1.06 and 1.04 (ω), respectively. Using the docking tool (CB-dock2), the two α-Syn-peptidomimetic inhibitor complexes (α-Syn-NPT100-18A and α-Syn-NPT200-11) have been prepared. Then all-atom molecular dynamics (MD) simulation was carried out on the α-Syn (control), α-Syn-NPT100-18A and α-Syn-NPT200-11 complex systems in presence of DOPE: DOPS: DOPC (5:3:2) lipid bilayer. From the conformational dynamics analysis, the 3-D structure of α-Syn was found to be stable, and the helices present in the regions (1-37) and (45-95) of α-Syn were found to be retained in the presence of the two peptidomimetic inhibitors. The electron density profile analysis revealed the binding modes of NAC and C-terminal region of α-Syn (in the presence of NPT200-11 inhibitor) with lipid membrane are in the close vicinity from the lipid bilayer centre. Our findings in this study on α-Syn-membrane interactions may be useful for developing a new therapeutic approach for treating PD and other neurodegenerative disorders.Communicated by Ramaswamy H. Sarma.

Structural analysis of human ATE1 isoforms and their interactions with Arg-tRNAArg.

Posttranslational protein arginylation has been shown as a key regulator of cellular processes in eukaryotes by affecting protein stability, function, and interaction with macromolecules. Thus, the enzyme Arginyltransferase and its targets, are of immense interest to modulate cellular processes in the normal and diseased state. While the study on the effect of this posttranslational modification in mammalian systems gained momentum in the recent times, the detail structures of human ATE1 (hATE1) enzymes has not been investigated so far. Thus, the purpose of this study was to predict the overall structure and the structure function relationship of hATE1 enzyme and its four isoforms. The structure of four ATE1 isoforms were modelled and were docked with 3'end of the Arg-tRNAArg which acts as arginine donor in the arginylation reaction, followed by MD simulation. All the isoforms showed two distinct domains. A compact domain and a somewhat flexible domain as observed in the RMSF plot. A distinct similarity in the overall structure and interacting residues were observed between hATE1-1 and X4 compared to hATE1-2 and 5. While the putative active sites of all the hATE1 isoforms were located at the same pocket, differences were observed in the active site residues across hATE1 isoforms suggesting different substrate specificity. Mining of nsSNPs showed several nsSNPs including cancer associated SNPs with deleterious consequences on hATE1 structure and function. Thus, the current study for the first time shows the structural differences in the mammalian ATE1 isoforms and their possible implications in the function of these proteins.Communicated by Ramaswamy H. Sarma.

Identification of FDA-approved drugs with triple targeting mode of action for the treatment of monkeypox: a high throughput virtual screening study.

According to the Center for Disease Control and Prevention, as of August 23, 94 countries had confirmed 42,954 Monkeypox Virus cases. As specific monkeypox drugs are not yet developed, the treatment depends on repurposed FDA-approved drugs. According to a recent study, the Monkeypox outbreak is caused by a strain with a unique mutation, raising the likelihood that the virus will develop resistance to current drugs by acquiring mutations in the targets of currently used drugs. The probability of multiple mutations in two or more drug targets at a time is always low than mutation in a single drug target. Therefore, we identified 15 triple-targeting FDA-approved drugs that can inhibit three viral targets, including topoisomerase1, p37, and thymidylate kinase, using high throughput virtual screening approach. Further, the molecular dynamics simulation analysis of the top hits such as Naldemedine and Saquinavir with their respective targets reveals the formation of stable conformational changes of the ligand-protein complexes inside the dynamic biological environment. We suggest further research on these triple-targeting molecules to develop an effective therapy for the currently spreading Monkeypox.

Conformational dynamics of A30G α-synuclein that causes familial Parkinson disease.

The first gene shown to be responsible for autosomal-dominant Parkinson's disease (PD) is the SNCA gene, which encodes for alpha synuclein (α-Syn). Recently, a novel heterozygous A30G mutation of the SNCA gene associated with familial PD has been reported. However, little research has been done on how the A30G mutation affects the structure of α-Syn. So, using atomistic molecular dynamics (MD) simulation, we demonstrate here the key structural characteristics of A30G α-Syn in the free monomer form and membrane associated state. From the MD trajectory analysis, the structure of A30G α-Syn was noticed to exhibit rapid conformational change, increase in backbone flexibility near the site of mutation and decrease in α-helical propensity. The typical torsion angles in residues (Val26 and Glu28) near the mutation site were observed to deviate significantly in A30G α-Syn. In the case of membrane bound A30G α-Syn, the regions that were submerged in the lipid bilayer (N-helix (3-37) and turn region (38-44)) found to contain higher helical content than the elevated region above the lipid surface. The bending angle in the helix-N and helix-C regions were noticed to be relatively higher in the free form of A30G α-Syn (38.50) than in the membrane bound form (370). The A30G mutation in α-Syn was predicted to have an impact on the stability and function of the protein based on ΔΔG values obtained from the online servers. Our results demonstrate that the A30G mutation in α-Syn altered the protein's α-helical structure and slightly altered the membrane binding.Communicated by Ramaswamy H. Sarma.

Chemical system biology approach to identify multi-targeting FDA inhibitors for treating COVID-19 and associated health complications.

In view of many European countries and the USA leading to the second wave of COVID-19 pandemic, winter season, the evolution of new mutations in the spike protein, and no registered drugs and vaccines for COVID-19 treatment, the discovery of effective and novel therapeutic agents is urgently required. The degrees and frequencies of COVID-19 clinical complications are related to uncontrolled immune responses, secondary bacterial infections, diabetes, cardiovascular disease, hypertension, and chronic pulmonary diseases. It is essential to recognize that the drug repurposing strategy so far remains the only means to manage the disease burden of COVID-19. Despite some success of using single-target drugs in treating the disease, it is beyond suspicion that the virus will acquire drug resistance by acquiring mutations in the drug target. The possible synergistic inhibition of drug efficacy due to drug-drug interaction cannot be avoided while treating COVID-19 and allied clinical complications. Hence, to avoid the unintended development drug resistance and loss of efficacy due to drug-drug interaction, multi-target drugs can be promising tools for the most challenging disease. In the present work, we have carried out molecular docking studies of compounds from the FDA approved drug library, and the FDA approved and passed phase -1 drug libraries with ten therapeutic targets of COVID-19. Results showed that known drugs, including nine anti-inflammatory compounds, four antibiotics, six antidiabetic compounds, and one cardioprotective compound, could effectively inhibit multiple therapeutic targets of COVID-19. Further in-vitro, in vivo, and clinical studies will guide these drugs' proper allocation to treat COVID-19.Communicated by Ramaswamy H. Sarma.

Computational investigation on the effect of Oleuropein aglycone on the α-synuclein aggregation.

Parkinson's disease (PD) is considered to be the second most common progressive neurodegenerative brain disorder after Alzheimer's disease, which is caused by misfolding and aggregation of Alpha-synuclein (α-synuclein). It is characterized by distinct aggregated fibrillary form of α-synuclein known as the Lewy bodies and Lewy neurites. The most promising approach to combat PD is to prevent the misfolding and subsequent aggregation of α-synuclein. Recently, Oleuropein aglycone (OleA) has been reported to stabilize the monomeric structure of α-synuclein, subsequently favoring the growth of nontoxic aggregates. Therefore, understanding the conformational dynamics of α-synuclein monomer in the presence of OleA is significant. Here, we have investigated the effect of OleA on the conformational dynamics and the aggregation propensity of α-synuclein using molecular dynamics simulation. From molecular dynamics trajectory analysis, we noticed that when OleA is bound to α-synuclein, the intramolecular distance between non-amyloid-ß component domain and C-terminal domain of α-synuclein was increased, whereas long-range hydrophobic interactions between the two region were reduced. Oleuropein aglycone was found to interact with the N-terminal domain of α-synuclein, making this region unavailable for interaction with membranes and lipids for the formation of cellular toxic aggregates. From the binding-free energy analysis, we found binding affinity between α-synuclein and OleA to be indeed high (ΔGbind = -12.56 kcal mol-1 from MM-PBSA and ΔGbind = -27.41 kcal mol-1from MM-GBSA). Our findings in this study thus substantiate the effect of OleA on the structure and stabilization of α-synuclein monomer that subsequently favors the growth of stable and nontoxic aggregates.Communicated by Ramaswamy H. Sarma.

Effect of CTerm of human albumin on the aggregation propensity of Aß1-42 peptide: a potential of mean force study.

Alzheimer's disease (AD) is the most common progressive neurodegenerative brain disorder. It is characterized by the presence of extracellular aggregated fibrillary form of amyloid beta (Aß) peptide and intraneuronal neurofibrillary tangles caused by the hyperphosphorylation of tau protein. Monomeric form of Aß peptide in α-conformation is not toxic but it can undergo self-aggregation to form ß-conformation which is neurotoxic. The most promising approach to combat AD is to prevent the self-aggregation of Aß peptide. Recently, it has been reported that C-terminal (CTerm) of human albumin (HA) binds to the Aß1-42 peptide and impairs the Aß1-42 aggregation and promotes disassembly of Aß1-42 aggregates. In this work, using potential of mean force (PMF) and binding free energy (BFE) calculations, we have demonstrated the effect of CTerm of HA on the dimerization of Aß1-42 peptide. From the PMF profile, we noticed Aß1-42-CTerm Heterodimer (10.99 kcal mol - 1) complex to have higher disassociation energy than Aß1-42-Aß1-42 homodimer (2.23 kcal mol - 1) complex. And also from the BFE calculations, we found that the binding affinity between Aß1-42 peptide and CTerm (ΔGbind = -32.27 kcal mol - 1 from MM-GBSA and ΔGbind = -2.83 kcal mol - 1 from MM-PBSA (molecular mechanics-Poisson - Boltzmann surface area)) to be stronger than the Aß1-42 peptide and another Aß1-42 peptide (ΔGbind = -16.20 kcal mol - 1 from MM-GBSA and ΔGbind = -1.95 kcal mol - 1 from MM-PBSA). In this study, our findings from PMF and BFE analysis of the two complexes provide salient structural, binding and unbinding features and thermodynamics that support the ability of CTerm of HA in affecting the dimerization of Aß1-42. Communicated by Ramaswamy H. Sarma.

Computational Investigation on the p53-MDM2 Interaction Using the Potential of Mean Force Study.

Murine double minute 2 (MDM2) proteins are found to be overproduced by many human tumors in order to inhibit the functioning of p53 molecules, a tumor suppressor protein. Thus, reactivating p53 functioning in cancer cells by disrupting p53-MDM2 interactions may offer a significant approach in cancer treatment. However, the structural characterization of the p53-MDM2 complex at the atomistic level and the mechanism of binding/unbinding of the p53-MDM2 complex still remain unclear. Therefore, we demonstrate here the probable binding (unbinding) pathway of transactivation domain 1 of p53 during the formation (dissociation) of the p53-MDM2 complex in terms of free energy as a function of reaction coordinate from the potential of mean force (PMF) study using two different force fields: ff99SB and ff99SB-ILDN. From the PMF plot, we noticed the PMF to have a minimum value at a p53-MDM2 separation of 12 Å, with a dissociation energy of 30 kcal mol-1. We also analyzed the conformational dynamics and stability of p53 as a function of its distance of separation from MDM2. The secondary structure content (helix and turns) in p53 was found to vary with its distance of separation from MDM2. The p53-MDM2 complex structure with lowest potential energy was isolated from the ensemble at the reaction coordinate corresponding to the minimum PMF value and subjected to molecular dynamics simulation to identify the interface surface area, interacting residues at the interface, and the stability of the complex. The simulation results highlight the importance of hydrogen bonds and the salt bridge between Lys94 of MDM2 and Glu17 of p53 in the stability of the p53-MDM2 complex. We also carried out the binding free energy calculations and the per residue energy decomposition analyses of the interface residues of the p53-MDM2 complex. We found that the binding affinity between MDM2 and p53 is indeed high [ΔG bind = -7.29 kcal mol-1 from molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) and ΔG bind = -53.29 kcal mol-1 from molecular mechanics/generalized borne surface area]. The total binding energy obtained using the MM/PBSA method was noticed to be closer to the experimental values (-6.4 to -9.0 kcal mol-1). The p53-MDM2 complex binding profile was observed to follow the same trend even in the duplicate simulation run and also in the simulation carried out with different force fields. We found that Lys51, Leu54, Tyr100, and Tyr104 from MDM2 and the residues Phe19, Trp23, and Leu26 from p53 provide the highest energy contributions for the p53-MDM2 interaction. Our findings highlight the prominent structural and binding characteristics of the p53-MDM2 complex that may be useful in designing potential inhibitors to disrupt the p53-MDM2 interactions.

1α, 25-dihydroxy Vitamin D3 containing fractions of Catharanthus roseus leaf aqueous extract inhibit preadipocyte differentiation and induce lipolysis in 3T3-L1 cells.

BACKGROUND: To investigate the potential of Catharanthus roseus leaf aqueous crude extract (CRACE) as a regulator of adipocyte development and function. METHODS: 3T3-L1 adipogenesis model was used to investigate the effect of CRACE on adipogenesis. 3T3-L1 preadipocytes (for adipogenic differentiation) and mature 3T3-L1 adipocytes (for adipocyte function) were treated with non-toxic doses of CRACE. The outcomes were corroborated by intracellular lipid accumulation, expression of pro-and anti-adipogenic effector molecules. To investigate CRACE mediated lipolysis, cAMP accumulation, glycerol release and phosphorylation of key effector molecules were tested in treated mature adipocytes. Finally, the extract was fractionated to identify the active molecule/s in the extract. RESULTS: CRACE significantly reduced adipocyte differentiation by modulating PPARγ expression. At early stage CRACE directly targeted Lipin1 expression and consequently impacted KLF7, subsequently expression of GATA2, CEBPα, SREBP1c were targeted, with PPARγ expression, particularly curtailed. While CRACE significantly reduced several lipogenic genes like FAS and GPD1 in mature adipocytes, concomitantly, it greatly increased lipolysis resulting in decreased lipid accumulation in mature adipocytes. The increase in lipolysis was due to decreased Akt activation, increased cAMP level, and PKA activity. The fractionation of CRACE allowed identification of two fractions with potent anti-adipogenic activity. Both the fractions contained 1α, 25-dihydroxy Vitamin D3 as major component. CONCLUSIONS: 1α, 25-dihydroxy Vitamin D3 containing CRACE can be developed into an effective anti-obesity formulation that decreases adipogenesis and increases lipid catabolism.

Consequences of Heterogeneous Crowding on an Enzymatic Reaction: A Residence Time Monte Carlo Approach.

Translational diffusion of a free substrate in crowded metabolically active spaces such as cell cytoplasm or mitochondrial matrix is punctuated by collisions and nonspecific interactions with soluble/immobile macromolecules/macrostructures in a variety of shapes/sizes. It is not understood how such disruptions alter enzyme reaction kinetics in such spaces. A novel Monte Carlo (MC) technique, "residence time MC", has been developed to study the kinetics of a simple enzyme-substrate reaction in a crowded milieu using a single immobile enzyme in the midst of diffusing substrates and products. The reaction time lost while the substrate nonspecifically interacts or is transiently trapped with ambient macromolecules is quantified by introducing the residence time "tau". Tau scales with the size of crowding macromolecules but makes the knowledge of their shape redundant. The residence time thus presents a convenient parameter to realistically mimic the sticky surroundings encountered by a diffusing substrate in heterogeneously crowded physiological spaces. Results reveal that for identical substrate concentration and excluded volume, increase in tau significantly diminished enzymatic product yield and reaction rate, slowed down substrate/product diffusion, and prolonged their relaxation times. A smooth transition from the anomalous subdiffusive motion to normal diffusion at long time limits was observed irrespective of the value of tau. The predictions from the model are shown to be in qualitative agreement with in vitro experimental data revealing the rate of alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate in the midst of 40/500/2000 kDa dextrans. Our findings from the residence time MC model also attempt to rationalize previously unexplained experimental observations in crowded enzyme kinetics literature. Furthermore, major insights to emerge from this study are the reasons why free diffusion of the substrate in crowded physiological spaces is detrimental to enzyme function. It is argued that organized enzyme clusters such as "metabolon" may perhaps exist to regulate the substrate translocation in such sticky physiological spaces to maintain optimal enzyme function. In summary, this work provides key insights explaining why absence of substrate channeling can dramatically slow down enzyme reaction rate in crowded metabolically active spaces.

Investigation of the probable homo-dimer model of the Xeroderma pigmentosum complementation group A (XPA) protein to represent the DNA-binding core.

The Xeroderma pigmentosum complementation group A (XPA) protein functions as a primary damage verifier and as a scaffold protein in nucleotide excision repair (NER) in all higher organisms. New evidence of XPA's existence as a dimer and the redefinition of its DNA-binding domain (DBD) raises new questions regarding the stability and functional position of XPA in NER. Here, we have investigated XPA's dimeric status with respect to its previously defined DBD (XPA98-219) as well as with its redefined DBD (XPA98-239). We studied the stability of XPA98-210 and XPA98-239 homo-dimer systems using all-atom molecular dynamics simulation, and we have also characterized the protein-protein interactions (PPI) of these two homo-dimeric forms of XPA. After conducting the root mean square deviation (RMSD) analyses, it was observed that the XPA98-239 homo-dimer has better stability than XPA98-210. It was also found that XPA98-239 has a larger number of hydrogen bonds, salt bridges, and hydrophobic interactions than the XPA98-210 homo-dimer. We further found that Lys, Glu, Gln, Asn, and Arg residues shared the major contribution toward the intermolecular interactions in XPA homo-dimers. The binding free energy (BFE) analysis, which used the molecular mechanics Poisson-Boltzmann method (MM-PBSA) and the generalized Born and surface area continuum solvation model (GBSA) for both XPA homo-dimers, also substantiated the positive result in favor of the stability of the XPA98-239 homo-dimer. Communicated by Ramaswamy H. Sarma.

Structure-Based Virtual Screening of High-Affinity ATP-Competitive Inhibitors Against Human Lemur Tyrosine Kinase-3 (LMTK3) Domain: A Novel Therapeutic Target for Breast Cancer.

Human lemur tyrosine kinase-3 (LMTK3) is an oncogenic kinase known to regulate ER-α through phosphorylation and is considered to be a novel therapeutic target for breast cancer. In this work, we have studied the ATP-binding mechanism with LMTK3 domain and also carried out virtual screening on LMTK3 to identify lead compounds using Dock blaster server. The top scored compounds obtained from Dock blaster were then narrowed down further to six lead compounds (ZINC37996511, ZINC83363046, ZINC3745998, ZINC50456700, ZINC83351792 and ZINC83364581) based on high-binding affinity and non-bonding interactions with LMTK3 using Autodock 4.2 program. We found in comparison to ATP, the lead compounds bind relatively stronger to LMTK3. The relative binding free energy results from MM-PBSA/GBSA method further indicate the strong binding affinity of lead compounds over ATP to LMTK3 in the dynamic system. Further, potential of mean force (PMF) study for ATP and lead compounds with LMTK3 have been performed to explore the unbinding processes and the free energy barrier. From the PMF results, we observed that the lead compounds have higher dissociation energy barriers than the ATP. Our findings suggest that these lead compounds may compete with ATP, and could act as probable potential inhibitors for LMTK3.

Characterizing the Binding Interactions between DNA-Binding Proteins, XPA and XPE: A Molecular Dynamics Approach.

The scaffold nature of Xeroderma pigmentosum complementation group A (XPA) protein makes it an important member of nucleotide excision repair (NER) that removes bulky DNA lesions with the help of various protein-protein interactions (PPI) and DNA-protein interactions. However, many structural insights of XPA's interaction and the binding patterns with other NER proteins are yet to be understood. Here, we have studied one such crucial PPI of XPA with another NER protein, Xeroderma pigmentosum complementation group A (XPE), by using the previously identified binding site of XPA (residues 185-226) in the Assisted Model Building With Energy Refinement force-field-mediated dynamic system. We studied the relationship between XPA185-226-XPE complex using three different docked models. The major residues observed in all of the models that were responsible for the PPI of this complex were Arg20, Arg47, Asp51, and Leu57 from XPE and the residues Leu191, Gln192, Val193, Trp194, Glu198, Glu202, Glu205, Arg207, Glu209, Gln216, and Phe219 from XPE185-226. During the simulation study, the orientation of XPA was also noted to be changed by almost 180° in models 1 and 3, which remain unchanged in model 2, indicating that XPA interacts with XPE with its N-terminal end facing downward and C-terminal end facing upward. The same was concurrent with the binding of DNA-binding domain region of XPA (aa98-239) with XPE. The N-terminal of XPE was stretched for accommodating XPA. Using the per-residue energy decomposition analysis for the interface residues of all models, the binding affinity between these proteins were found to be dependent on R20, R47, and L57 of XPE and the residues L191, V193, W194, E198, E202, E205, R207, and F219 of XPA. The net binding free energy of the XPA185-226-XPE protein complex was found to be -48.3718 kcal mol-1 for model 1, -49.09 kcal mol-1 for model 2, and -56.51 kcal mol-1 for model 3.

Structural dynamics and interactions of Xeroderma pigmentosum complementation group A (XPA98-210) with damaged DNA.

Nucleotide excision repair (NER) in higher organisms repair massive DNA abrasions caused by ultraviolet rays, and various mutagens, where Xeroderma pigmentosum group A (XPA) protein is known to be involved in damage recognition step. Any mutations in XPA cause classical Xeroderma pigmentosum disease. The extent to which XPA is required in the NER is still unclear. Here, we present the comparative study on the structural and conformational changes in globular DNA binding domain of XPA98-210 in DNA bound and DNA free state. Atomistic molecular dynamics simulation was carried out for both XPA98-210 systems using AMBER force fields. We observed that XPA98-210 in presence of damaged DNA exhibited more structural changes compared to XPA98-210 in its free form. When XPA is in contact with DNA, we found marked stability of the complex due to the formation of characteristic longer antiparallel ß-sheets consisting mainly lysine residues.

In silico investigation on the inhibition of Aß42 aggregation by Aß40 peptide by potential of mean force study.

Recent experimental data revealed that small, soluble Amyloid beta (Aß42) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer's disease. Here, we have studied dimerization of Aß42/Aß42 homo-dimer and Aß40/Aß42 hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aß40 peptide, there exists a strong tendency to form a hetero-dimer with Aß42 peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aß40 on the Aß42 peptide. Our study also shows that in presence of Aß40, the beta-content of Aß42 monomer is reduced. Additionally, certain residues important for bending in Aß42 peptide attained an increased flexibility in the presence of Aß40. The salt-bridge destabilization also manifested the impact of Aß40 on Aß42 peptide as a whole. Based on this, one may expect that Aß40 inhibits the aggregation propensity of Aß42. Moreover, the binding free energy obtained by the molecular mechanics-Poisson-Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aß40 binding induces conformational change in Aß42. Our results suggest that co-oligomerization of Aß isoforms may play a substantial role in Alzheimer's disease.

Computational investigation on the effects of H50Q and G51D mutations on the α-Synuclein aggregation propensity.

The aggregation of α-synuclein is linked directly to the histopathology of Parkinson's disease (PD). However, several missense mutations present in the α-synuclein gene (SNCA) have been known to be associated with PD. Several studies have highlighted the effect of SNCA mutations on the α-synuclein aggregation, but their pathological roles are not completely established. In this study, we have focused on the effects of the recently discovered α-synuclein missense mutants (H50Q and G51D) on the aggregation using computational approaches. We performed all atom molecular dynamics (MD) simulation on these mutants and compared their conformational dynamics with Wild-Type (WT) α-synuclein. We noticed the solvent accessible surface area (SASA), radius of gyration, atomic fluctuations, and beta strand content to be higher in H50Q than G51D and WT. Using PDBSum online server; we analyzed the inter-molecular interactions that drive the association of monomeric units of H50Q, WT, and G51D in forming the respective homo-dimer. We noticed the interface area, number of interacting residues and binding free energy to be higher for H50Q homo-dimer than the WT and G51D homo-dimers. Our findings in this study suggest that in comparison to WT and G51D, H50Q mutation to have a positive effect on increasing the α-synuclein aggregation propensity. Hence, we see that H50Q and G51D mutation show conflicting effect on the aggregation propensity of α-synuclein.

Structural Characterization of Amyloid ß17-42 Dimer by Potential of Mean Force Analysis: Insights from Molecular Dynamics Simulations.

BACKGROUND: Recent experiments with Amyloid ß1-42 peptide have indicated that the initial dimerization of Aß1-42 monomers to form amyloid dimers stand out as a key event in the generation of toxic oligomers. However, the structural characterization of Aß1-42 dimer at the atomistic level and the dimerization mechanism by which Aß1-42 peptides co-aggregate still remains not clear. OBJECTIVE: In the present study, the process of Aß17-42 peptide dimerization which is known to play an important role in the plaque formation in Alzheimer's disease was evaluated in terms of potential of mean force. METHODS: The Aß17-42 dimer was constructed using PatchDock server. We have used molecular dynamics (MD) simulation with the umbrella sampling methodology to compute the Potential of Mean Force for the dimerization of Aß17-42. The global minima structure at the minimum distance of separation was isolated from the calculated free energy profile and the interactions involved in the formation of the dimer structure were examined. Protein-protein interfaces and the residueresidue interactions vital for generation of the dimer complexes were also evaluated. RESULTS: The simulation results elucidated the interaction between the monomeric units to be governed primarily by the hydrophobic and hydrogen bonds. The resultant Aß17-42 dimer was found to have an increased ß-strands propensity at the hydrophobic regions encompassing the CHC region. Furthermore, specific hydrophobic residues were found to play a vital role in the formation of the dimer complex. CONCLUSION: From the results we may therefore conclude hydrophobic region encompassing the CHC region to be crucial in dimerization process. The findings from this study provide detailed information for the complex process of early events of Aß aggregation.

An in silico approach to understand the structure-function properties of a serine protease (Bacifrinase) from Bacillus cereus and experimental evidence to support the interaction of Bacifrinase with fibrinogen and thrombin.

Microbial fibrinogenolytic serine proteases find therapeutic applications in the treatment of thrombosis- and hyperfibrinogenemia-associated disorders. However, analysis of structure-function properties of an enzyme is utmost important before its commercial application. In this study, an attempt has been made to understand the structure of a fibrinogenolytic protease enzyme, "Bacifrinase" from Bacillus cereus strain AB01. From the molecular dynamics trajectory analysis, the modelled three-dimensional structure of the protease was found to be stable and the presence of a catalytic triad made up of Asp102, His83 and Ser195 suggests that it is a serine protease. To understand the mechanism of enzyme-substrate and enzyme-inhibitor interactions, the equilibrated protein was docked with human fibrinogen (the physiological substrate of this enzyme), human thrombin and with ten selective protease inhibitors. The Bacifrinase-chymostatin interaction was the strongest among the selected protease inhibitors. The serine protease inhibitor phenyl methane sulphonyl fluoride was found to interact with the Ser134 residue of Bacifrinase. Furthermore, protein-protein docking study revealed the fibrinogenolytic property of Bacifrinase and its interaction with Aα-, Bß- and Cγ-chains human fibrinogen to a different extent. However, biochemical analysis showed that Bacifrinase did not hydrolyse the γ-chain of fibrinogen. The in silico and spectrofluorometric studies also showed interaction of Bacifrinase with thrombin as well as fibrinogen with a Kd value of 16.5 and .81 nM, respectively. Our findings have shed light on the salient structural features of Bacifrinase and confirm that it is a fibrinogenolytic serine protease.