Effect of osmolytes on the EcoRI endonuclease: Insights into hydration and protein dynamics from molecular dynamics simulations.

Osmolytes play an important role in cellular physiology by modulating the properties of proteins, including their molecular specificity. EcoRI is a model restriction enzyme whose specificity to DNA is altered in the presence of osmolytes. Here, we investigate the effect of two different osmolytes, glycerol and DMSO, on the dynamics and hydration of the EcoRI enzyme using molecular dynamics simulations. Our results show that the osmolytes, alter the essential dynamics of EcoRI. Particularly, we observe that the dynamics of the arm region of EcoRI which is involved in DNA binding is significantly altered. In addition, conformational free energy analyses reveals that the osmolytes bring about a change in the landscape similar to that of EcoRI bound to cognate DNA. We further observe that the hydration of the enzyme for each of the osmolyte is different, indicating that the mechanism of action of each of these osmolytes could be different. Further analyses of interfacial water dynamics using rotational autocorrelation function reveals that while the protein surface contributes to a slower tumbling motion of water, osmolytes, additionally contribute to the slowing of the angular motion of the water molecules. Entropy analysis also corroborates with this finding. We also find that the slowed rotational motion of interfacial waters in the presence of osmolytes contributes to a slowed relaxation of the hydrogen bonds between the interfacial waters and the functionally important residues in the protein. Taken together, our results show that osmolytes alter the dynamics of the protein by altering the dynamics of water. This altered dynamics, mediated by the changes in the water dynamics and hydrogen bonds with functionally important residues, may contribute to the altered specificity of EcoRI in the presence of osmolytes.

Comprehensive screening of marine metabolites against class B1 metallo-ß-lactamases of Klebsiella pneumoniae using two-pronged in silico approach.

The emergence of antibiotic resistance is one of the major global threats in healthcare. Metallo-ß-Lactamases (MBL) are a class of enzymes in bacteria that cleave ß-lactam antibiotics and confer resistance. MBLs are further divided into subclasses B1, B2 and B3. Of these, subclasses B1-MBLs (including NDM-1, VIM-2 and IMP-1) constitute the clinically prevalent lactamases conferring resistance. To date, no effective drugs are available clinically against MBLs. In this work, we aim to identify potent inhibitors for the B1 subclass of MBL from available marine metabolites in Comprehensive Marine Natural Product database through integrated in silico approaches. We have used two methods, namely, the high-throughput strategy and the pharmacophore-based strategy to identify potential inhibitors from marine metabolites. High-throughput virtual screening identified N-methyl mycosporine-Ser, which had the highest binding affinity to NDM-1. The pharmacophore-based approach based on co-crystallized ligands identified makaluvic acid and didymellamide with higher binding affinity across B1-MBLs. Taking into account of the advantage of a pharmacophore model-based approach with higher binding affinity, we conclude that both makaluvic acid and didymellamide show potential broad-spectrum effects by binding to all three B1-MBL receptors. The study also indicates the need to take multiple in silico approaches to screen and identify novel inhibitors. Together, our study reveals promising inhibitors that can be identified from marine systems.Communicated by Ramaswamy H. Sarma.

Effect of glycerol on free DNA: A molecular dynamics simulation study.

Osmolytes are a class of organic solutes that are produced in a variety of organisms in response to stress. They exert diverse effects on macromolecules and their functions. In this work, we investigate the effect of glycerol, one such osmolyte, on the hydration and conformation of four DNA sequences that differ by a single base pair and a random DNA sequence. Molecular dynamics simulations reveal DNA sequence-dependent and glycerol concentration-dependent hydration and DNA conformation. Interestingly, we find that the sequence-dependent changes in the hydration reflects the order of preference of these sequences for star activity of the EcoRI enzyme. However, the changes in DNA conformation do not reflect this order of preference. Interaction energy analyses reveal that the per-glycerol interaction energy with DNA is stronger than the per-water interaction energy with DNA. However, the total interaction energy of glycerol with DNA is lower than that of total water-DNA interaction energy indicating that it might be easier for an approaching DNA-binding protein to displace glycerol than water and thus contributing positively to protein-DNA binding. In a larger context, our study brings attention to the need to investigate the effect of osmolytes on free DNA in order to delineate the role of osmolyte in protein-DNA interactions.

In vitro and in silico screening of Klebsiella pneumoniae new Delhi metallo-ß-lactamase-1 inhibitors from endophytic Streptomyces spp.

The increase in drug resistance over the last two decades is a big threat in health care settings. More importantly, the dissemination of carbapenem-resistant Enterobacteriaceae is the major threat to public health with an increase in morbidity and mortality. ß-lactamase is known to confer enteric bacteria with nearly complete resistance to all ß-lactam antibiotics including the late-generation carbapenems. The commercially available ß-lactamase inhibitors, clavulanic acid, sulbactam, and tazobactam are being met with an increasing number of resistant phenotypes and are ineffective against pathogens harbouring New Delhi metallo-ß-lactamase (NDM-1). Inhibition of New Delhi metallo-ß-lactamase-1 activity is one potential way to treat metallo ß-lactamase (MBL) producing multi drug resistant (MDR) pathogen. The present study focused on screening of Klebsiella pneumoniae New Delhi metallo-ß-lactamase-1 (BLIs) from endophytic Streptomyces spp. using in vitro and in silico methods. The study identified three potential inhibitors of New Delhi metallo-ß-lactamase-1, namely dodecanoic acid, dl-alanyl-l-leucine and phenyl propanedioic acid. These molecules were found to bind to other MBLs namely, IMP-1 and VIM-2. To the best of our knowledge, this is the first kind of study reporting the binding mode of these molecules with New Delhi metallo-ß-lactamase-1.Communicated by Ramaswamy H. Sarma.