Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

<div><p>In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.</p></div

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation

<p>Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in <i>Drosophila melanogaster</i> are known to bind cooperatively to 5′-(T/C)TAATTAA(T/A)(T/A)G-3′ DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein–DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll–3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll–3A01–Al. In addition, this change in secondary structure of Cll does not allow it to form protein–protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al–3A01–Cll where Cll binds to Al–3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein–protein interactions while proposing likely mechanism of recognition in ternary complex, Al–Cll–DNA.</p

An Escherichia coli strain, PGB01, isolated from feral pigeon Faeces, thermally fit to survive in pigeon, shows high level resistance to trimethoprim.

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon's gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr

Exploring GPR109A Receptor Interaction with Hippuric Acid Using MD Simulations and CD Spectroscopy

Previous research has indicated that various metabolites belonging to phenolic acids (PAs), produced by gut microflora through the breakdown of polyphenols, help in promoting bone development and protecting bone from degeneration. Results have also suggested that G-protein-coupled receptor 109A (GPR109A) functions as a receptor for those specific PAs such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA). Indeed, HA has a molecular structural similarity with nicotinic acid (niacin) which has been shown previously to bind to GPR109A receptor and to mediate antilipolytic effects; however, the binding pocket and the structural nature of the interaction remain to be recognized. In the present study, we employed a computational strategy to elucidate the molecular structural determinants of HA binding to GPR109A and GPR109B homology models in understanding the regulation of osteoclastogenesis. Based on the docking and molecular dynamics simulation studies, HA binds to GPR109A similarly to niacin. Specifically, the transmembrane helices 3, 4 and 6 (TMH3, TMH4 and TMH6) and Extracellular loop 1 and 2 (ECL1 and ECL2) residues of GRP109A; R111 (TMH3), K166 (TMH4), ECL2 residues; S178 and S179, and R251 (TMH6), and residues of GPR109B; Y87, Y86, S91 (ECL1) and C177 (ECL2) contribute for HA binding. Simulations and Molecular Mechanics Poisson-Boltzmann solvent accessible area (MM-PBSA) calculations reveal that HA has higher affinity for GPR109A than for GPR109B. Additionally, in silico mutation analysis of key residues have disrupted the binding and HA exited out from the GPR109A protein. Furthermore, measurements of time-resolved circular dichroism spectra revealed that there are no major conformational changes in the protein secondary structure on HA binding. Taken together, our findings suggest a mechanism of interaction of HA with both GPR109A and GPR109B receptors

Hydrogen bonding between ligand and protein active site residues.

<p>Dash line shows hydrogen bond between, trimethoprim (magenta) and protein active site residues (green), A) TMP-sensitive-Dfr-TMP complex, B) PGB01-DfrA7-TMP complex.</p

Effect of microwave radiation on cell viability.

<p>A. Thermal effect of microwave radiation: Temperature of LB vs. time of exposure; B. Viability of cells vs. time of microwave exposure.</p

Diagram of amplified class 1 integron.

<p>Schematic representation of class 1 integron amplicon of the strain PGB01 amplified with the Int₂F-3′CS primer pair. Numbers correspond to sequence positions in EMBL Ac. No. FN563072.</p

Superimposition of protein active site residues.

<p>Superimposed side chain representation of protein active site residues of TMP-sensitive-Dfr (magenta) and PGB01-DfrA7 (orange).</p