MolDy: molecular dynamics simulation made easy.

MOTIVATION: Molecular dynamics (MD) is a computational experiment that is crucial for understanding the structure of biological macro and micro molecules, their folding, and the inter-molecular interactions. Accurate knowledge of these structural features is the cornerstone in drug development and elucidating macromolecules functions. The open-source GROMACS biomolecular MD simulation program is recognized as a reliable and frequently used simulation program for its precision. However, the user requires expertise, and scripting skills to carrying out MD simulations. RESULTS: We have developed an end-to-end interactive MD simulation application, MolDy for Gromacs. This front-end application provides a customizable user interface integrated with the Python and Perl-based logical backend connecting the Linux shell and Gromacs software. The tool performs analysis and provides the user with simulation trajectories and graphical representations of relevant biophysical parameters. The advantages of MolDy are (i) user-friendly, does not requiring the researcher to have prior knowledge of Linux; (ii) easy installation by a single command; (iii) freely available for academic research; (iv) can run with minimum configuration of operating systems; (v) has valid default prefilled parameters for beginners, and at the same time provides scope for modifications for expert users. AVAILABILITY AND IMPLEMENTATION: MolDy is available freely as compressed source code files with user manual for installation and operation on GitHub: https://github.com/AIBResearchMolDy/Moldyv01.git and on https://aibresearch.com/innovations.

ECEL1 related distal arthrogryposis 5D in an Indian cohort-Report of recognizable musculoskeletal phenotype and a possible founder variant.

Distal arthrogryposis type 5D (DA5D) is clinically characterized by knee extension contractures, distal joint contractures, clubfoot, micrognathia, ptosis, and scoliosis. We report nine affected individuals from eight unrelated Indian families with DA5D. Although the overall musculoskeletal phenotype is not very distinct from other distal arthrogryposis, the presence of fixed knee extension contractures with or without scoliosis could be an important early pointer to DA5D. We also report a possible founder variant in ECEL1 along with four novel variants and further expand the genotypic spectrum of DA5D.

Structural modeling of Omicron spike protein and its complex with human ACE-2 receptor: Molecular basis for high transmissibility of the virus.

Omicron is a new variant of SARS-CoV-2, which is currently infecting people around the world. Spike glycoprotein, an important molecule in pathogenesis of infection has been modeled and the interaction of its Receptor Binding Domain with human ACE-receptor has been analysed by simulation studies. Structural analysis of Omicron spike glycoprotein shows the 30 mutations to be distributed over all domains of the trimeric protein, wherein the mutant residues are seen to be participating in higher number of intra-molecular interactions including two salt bridges emanating from mutant residues thereby stabilizing their conformation, as compared to wild type. Complex of Receptor Binding Domain (RBD) with human ACE-2 receptor shows seven mutations at interacting interface comprising of two ionic interactions, eight hydrogen bonds and seven Van der Waals interactions. The number and quality of these interactions along with other binding biophysical parameters suggests more potency of RBD domain to the receptor as compared to the wild type counterpart. Results of this study explains the high transmissibility of Omicron variant of SARS-CoV-2 that is currently observed across the world.

Structural insights into the transient closed conformation and pH dependent ATPase activity of S.Typhi GyraseB N- terminal domain.

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.

Sequence Variation in the Response Element Determines Binding by the Transcription Factor p73.

How the sequence of a response element affects the binding of a transcription factor and, ultimately, the differential rate of transcription of genes under its control is not well-understood. In the case of the p73 transcription factor, it binds to >200 response elements to trigger developmental, cell arrest, and apoptotic pathways. The p73 response elements match the 20 bp consensus sequence of the p53 response elements that are formed by two 10 bp half-sites, where each half-site is an inverted repeat of two 5 bp quarter-sites. Using sedimentation velocity and fluorescence anisotropy experiments, we studied how systematic variations in the sequence of a half-site response element modify the DNA binding affinity of the p73 DNA-binding domain. We observed that each nucleotide position in the response element has a different influence in determining the binding of the p73 DNA-binding domain. The cytosine in the fourth position of each quarter-site is the largest determinant of DNA binding, followed by the nucleotide in the fifth position, and last, the first three positions show a slight regulatory preference for purines. Together with previous structural and functional results, our data suggest a hierarchical model of binding in which some nucleotide positions in the response element are more important than others in determining the binding of the transcription factor.

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code.

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.

Structure of p73 DNA-binding domain tetramer modulates p73 transactivation.

The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.

Suppression of conformation-compromised mutants of Salmonella enterica serovar Typhimurium MelB.

The crystal structure of the Na(+)-coupled melibiose permease of Salmonella enterica serovar Typhimurium (MelBSt) demonstrates that MelB is a member of the major facilitator superfamily of transporters. Arg residues at positions 295, 141, and 363 are involved in interdomain interactions at the cytoplasmic side by governing three clusters of electrostatic/polar interactions. Insertion of (one at a time) Glu, Leu, Gln, or Cys at positions R295, R141, and R363, or Lys at position R295, inhibits active transport of melibiose to a level of 2 to 20% of the value for wild-type (WT) MelBSt, with little effect on binding affinities for both sugar and Na(+). Interestingly, a spontaneous suppressor, D35E (periplasmic end of helix I), was isolated from the R363Q MelBSt mutant. Introduction of the D35E mutation in each of the mutants at R295, R141 (except R141E), or R363 rescues melibiose transport to up to 91% of the WT value. Single-site mutations for the pair of D35 and R175 (periplasmic end of helix VI) were constructed by replacing Asp with Glu, Gln, or Cys and R175 with Gln, Asn, or Cys. All mutants with mutations at R175 are active, indicating that a positive charge at R175 is not necessary. Mutant D35E shows reduced transport; D35Q and D35C are nearly inactivated. Surprisingly, the D35Q mutation partially rescues both R141C and R295Q mutations. The data support the idea that Arg at position 295 and a positive charge at positions 141 and 363 are required for melibiose transport catalyzed by MelBSt, and their mutation inhibits conformational cycling, which is suppressed by a minor modification at the opposite side of the membrane.

Crystal structures of the DNA-binding domain tetramer of the p53 tumor suppressor family member p73 bound to different full-site response elements.

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

The acidic C-terminal tail of DNA Gyrase of Salmonella enterica serovar Typhi controls DNA relaxation in an acidic environment.

The intracellular bacteria, Salmonella Typhi adapts to acidic conditions in the host cell by resetting the chromosomal DNA topology majorly controlled by DNA Gyrase, a Type II topoisomerase. DNA Gyrase forms a heterodimer A2B2 complex, which manages the DNA supercoiling and relaxation in the cell. DNA relaxation forms a part of the regulatory mechanism to activate the transcription of genes required to survive under hostile conditions. Acid-induced stress attenuates the supercoiling activity of the DNA Gyrase, resulting in DNA relaxation. Salmonella DNA becomes relaxed as the bacteria adapt to the acidified intracellular environment. Despite comprehensive studies on DNA Gyrase, the mechanism to control supercoiling activity needs to be better understood. A loss in supercoiling activity in E. coli was observed upon deletion of the non-conserved acidic C-tail of Gyrase A subunit. Salmonella Gyrase also contains an acidic tail at the C-terminus of Gyrase A, where its deletion resulted in reduced supercoiling activity compared to wild-type Gyrase. Interestingly, we also found that wild-type Gyrase compromises supercoiling activity at acidic pH 2-3, thereby causing DNA relaxation. The absence of a C-tail displayed DNA supercoiling to some extent between pH 2-9. Hence, the C-tail of Gyrase A might be one of the controlling factors that cause DNA relaxation in Salmonella at acidic pH conditions. We propose that the presence of the C-tail of GyraseA causes acid-mediated inhibition of the negative supercoiling activity of Gyrase, resulting in relaxed DNA that attracts DNA-binding proteins for controlling the transcriptional response.

Structure-based identification of potential natural compound inhibitors targeting bacterial cytoskeleton protein FtsZ from Acinetobacter baumannii by computational studies.

Acinetobacter baumannii is one of the multi-drug-resistant pathogens responsible for hospital-acquired infections reported worldwide. Clinically it is challenging to treat these pathogens as they have developed resistance against the existing class of antibiotics. Hence, there is an urgent need to develop a new class of antibiotics against these pathogens to prevent the spread of infections and mortality. In Acinetobacter baumannii, the filamentous temperature-sensitive mutant Z protein polymerizes at the imminent division site to form a Z-ring at the mid-point of the cell and act as a scaffold to recruit other cell division proteins involved in orchestrating septum synthesis in bacteria. Perturbation in the assembly of FtsZ affects bacterial cell dynamics and survival. Hence, FtsZ has emerged as a new drug target in antibiotic discovery to identify compounds that inhibit bacterial cell division. In this study, we have performed a virtual screening of 30,000 compounds from the ZINC Biogenic natural compound library targeting the nucleotide-binding site of FtsZ from Acinetobacter baumannii. We have identified 8 new natural compounds with binding energy in the range of -8.66 to -6.953 kcal/mol and analyzed them by 200 ns molecular dynamics simulations. Out of these eight compounds, ZINC14708526 showed the best binding with relatively optimal drug-likeness and medicinal chemistry as a potent inhibitor of abFtsZ. Thus, the identified FtsZ inhibitor ZINC14708526 is a promising lead compound to develop potent antimicrobial agents against Acinetobacter baumannii infections.Communicated by Ramaswamy H. Sarma.

Comprehensive omics studies of p53 mutants in human cancer.

The p53 is the master regulator of the cell known for regulating a large array of cellular processes. Inactivation of p53 by missense mutations is one of the leading causes of cancer. Some of these mutations endow p53 with selective oncogenic functions to promote tumor progression. Due to the vast array of mutations found in p53, the experimental studies showing the role of different mutant p53 as an oncogene are also expanding. In this review, we discuss the oncogenic roles of different p53 mutants at the cellular level identified by multi-omics tools. We discuss some of the therapeutic studies to tackle p53 mutants and their downstream targets identified by omics. We also highlight the future prospective and scope of further studies of downstream p53 targets by omics.

Mechanism of apoptosis activation by Curcumin rescued mutant p53Y220C in human pancreatic cancer.

The mutant p53Y220C (mutp53Y220C) is frequently observed in numerous tumors, including pancreatic cancer. The mutation creates a crevice in the DNA binding core domain and makes p53 a thermally unstable non-functional protein that assists tumor progression and confers resistance to chemotherapeutic drugs. Restoring mutp53 function to its wild type by selectively targeting this crevice with small molecules is a pivotal strategy to promote apoptosis. In this study, we have shown through different biophysical and cell-based studies that curcumin binds and rescues mutp53Y220C to an active wild-type conformation and restores its apoptotic transcription function in BxPC-3-pancreatic cancer cells. In addition, the curcumin-rescued-p53Y220C (CRp53) showed significant hyperphosphorylation at Ser15, Ser20, and acetylation at Lys382 with an 8-fold increase in transcription activity in the BxPC-3 cell lines. We also observed that the active CRp53 escapes Mdm2-mediated proteasomal degradation and the majority of the proteins were localized inside the nucleus with an increased half-life and transcription restoration compared to untreated BxPC-3 cells. By label-free proteomics analysis, we observed that upon curcumin treatment almost 227 proteins were dysregulated with the majority of them being transcriptional targets of p53. Based on our studies, it reflects that apoptosis in pancreatic cancer cells is mediated by curcumin-rescued mutant p53Y220C.

A patient with POLA1 splice variant expands the yet evolving phenotype of Van Esch O'Driscoll syndrome.

Van Esch-O'Driscoll syndrome (VEODS) is a rare cause of syndromic X-linked intellectual disability characterised by short stature, microcephaly, variable degree of intellectual disability, and hypogonadotropic hypogonadism. To date, heterozygous hypomorphic variants in the gene encoding the DNA Polymerase α subunit, POLA1, have been observed in nine patients from five unrelated families with VEODS. We report a three-year-old child with VEODS having borderline intellectual disability due to a novel splice site variant causing exon 6 skipping and reduced POLA1 expression.

Structural analysis of COVID-19 spike protein in recognizing the ACE2 receptor of different mammalian species and its susceptibility to viral infection.

The pandemic COVID-19 was caused by a novel Coronavirus-2 (SARS-CoV-2) that infects humans through the binding of glycosylated SARS-CoV-2 spike 2 protein to the glycosylated ACE2 receptor. The spike 2 protein recognizes the N-terminal helices of the glycosylated metalloprotease domain in the human ACE2 receptor. To understand the susceptibility of animals for infection and transmission, we did sequence and structure-based molecular interaction analysis of 16 ACE2 receptors from different mammalian species with SARS-CoV-2 spike 2 receptor binding domain. Our comprehensive structure analysis revealed that the natural substitution of amino acid residues Gln24, His34, Phe40, Leu79 and Met82 in the N-terminal α1 and α2 helices of the ACE2 receptor results in loss of crucial network of hydrogen-bonded and hydrophobic interactions with receptor binding domain of SARS-CoV-2 spike protein. Another striking observation is the absence of N-glycosylation site Asn103 in all mammals and many species, lack more than one N-linked glycosylation site in the ACE2 receptor. Based on the loss of crucial interactions and the absence of N-linked glycosylation sites we categorized Felis catus, Equus caballus, Panthera tigris altaica, as highly susceptible while Oryctolagus cuniculus, Bos Tauras, Ovis aries and Capra hircus as moderately susceptible species for infection. Similarly, the E. asinus, Bubalus bubalis, Canis lupus familiaris, Ailuropoda melaleuca and Camelus dromedarius are categorized as low susceptible with Loxodonta Africana, Mus musculus, Sus scrofa and Rattus rattus as least susceptible species for SARS-CoV-2 infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02599-2.

Curcumin rescue p53Y220C in BxPC-3 pancreatic adenocarcinomas cell line: Evidence-based on computational, biophysical, and in vivo studies.

BACKGROUND: The p53, tumor suppressor protein is inactivated upon mutation in the DNA-binding domain and the non-functional protein leads to cancers. The p53Y220C is one of the most frequently observed mutations in p53 with a scope of rescuing the protein function using small molecules. METHODS: Using computational modeling, biophysical, and experimental cell-based studies we tried to understand the molecular basis of Curcumin as a potential small molecule to stabilize p53Y220C mutant and restore its function. The pancreatic adenocarcinomas BxPC-3 p53Y220C mutant cell line was used for cell-based assays to determine the therapeutic potential of Curcumin to restore mutant p53 to function like wild type. RESULTS: Our results showed that the Curcumin binds p53Y220C with Kd = 3.169 ± 0.257 µM and it increases the DNA binding affinity of the mutant by 4-fold with Kd = 851.29 ± 186.27 nM. By Fluorescence, CD, and IR spectroscopy, we could characterize the secondary structural changes and stabilization of the p53Y220C DNA binding domain upon Curcumin binding. By caspase-3 and Annexin V assays, we could demonstrate that Curcumin at 3 µM to 8 µM concentration could initiate p53 mediated apoptosis in BxPC-3 cell line. Based on our experimental studies, we propose a mechanism for the activation of ATM/Chk1 kinases pathways for apoptosis and/or G2/M cell cycle arrest in the BxPC-3 cell line mediated by functionally restored p53Y220C. CONCLUSION: The study indicated that the natural compound Curcumin could rescue mutant p53Y220C in BxPC-3 pancreatic adenocarcinomas cell line to function like wild-type and activate apoptotic pathways.

Structure of the novel 14kDa fragment of alpha-subunit of phycoerythrin from the starving cyanobacterium Phormidium tenue.

The rod-like phycobilisome (PBS) in cyanobacterium is the light-harvesting complex of phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). The orderly degradation of PBS was observed under starvation conditions. A 14 kDa truncated fragment of alpha-subunit of PE (F-alphaPE) was identified from the degraded product. F-alphaPE was purified to homogeneity, sequenced and crystallized. The merohedrally twinned crystals with a twinning factor of approximately 0.5 were obtained. The crystal structure of F-alphaPE was determined with molecular replacement method using detwinned data and refined to an R(cryst) factor of 23.2% (R(free)=27.6%). The structure consisted of two crystallographically independent molecules in the asymmetric unit. The two molecules were designated as molecules A and B with a buried area of 200 A(2) at the interface. The structure of F-alphaPE consists of seven alpha-helices A, B, E, F, F', G and H. The first 31N-terminal residues that fold into parallel alpha-helices X and Y in other PEs are not present in the amino acid sequence of F-alphaPE. Both molecules, A and B contain two chromophore ligands, PEB1 and PEB2 in each. These are covalently linked to the polypeptide chain through Cys82 and Cys139, respectively. The superimposition of C(alpha) tracings of molecules A and B shows an r.m.s. shift of 1.0 A indicating that the structures of two independent molecules are very similar. The degradation of phycobilisome proteins under starvation stress seems to occur to supplement the requirement of amino acids for protein synthesis and to reduce the absorption of light energy.

Structural and conformational behavior of MurE ligase from Salmonella enterica serovar Typhi at different temperature and pH conditions.

MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.

The pivot point arginines identified in the ß-pinwheel structure of C-terminal domain from Salmonella Typhi DNA Gyrase A subunit.

The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA - C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.

Structure model of ferrochelatase from Salmonella Typhi elucidating metalation mechanism.

A homology model of ferrochelatase (HemH), the heme biosynthesis terminal step enzyme from Salmonella Typhi was generated to understand the mechanism of metal insertion into protoporphyrin IX for heme biosynthesis. The overall fold of membrane associated ferrochelatase (StFc) from S. Typhi is similar to human and Yeast ferrochelatase than Bacillus subtilis, and Bacillus anthracis. An insertion of 16 amino acid residues in helical switch having hydrophobic patch proposed to interact with membrane lipids and in opening and closing of heme binding cleft. The sequence analysis and the docking study revealed that the protoporphyrin binding site in StFc has a crucial replacement of Tyr/Met to Leu13 unique in comparison to other known structures, where Tyr13 observed in B. subtilis/B. anthracis while Met76 in human/yeast play important role in holding protoporphyrin in optimal orientation for metalation. A sitting-a-top (SAT) complex mechanism for metalation is proposed with His194 and Glu264 lie at the bottom and Leu13 on the top of the porphyrin ring. In addition, an entry and exit mechanism is also proposed for protoporphyrin binding into cavity by opening and closing of helical switch using molecular dynamics simulation studies of Apo and heme complexed model structure of S. Typhi HemH.