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The human butyrylcholinesterase (BChE) activity is less than 1% in the serum of silent variant individuals of Vysya community in India. They are homozygous for a point mutation at codon 307 (CTT → CCT) resulting in the substitution of leucine 307 by proline. The reason for the disappearance of the protein in the serum has not been explicated till date. Based on this background, we performed molecular dynamics simulation to probe the structural stability of Indian variant (L307P) in comparison with wild and other BChE variants (D70G, E497V, V142M) having differential esterase activity. The simulation of all the mutants except D70G showed a much larger Cα root mean square deviation from the wild BChE crystal structure, showing the overall conformational disturbance. Further analysis revealed that secondary structure of the mutant proteins was not stable. The orientation of the catalytic triad is also distorted in all the mutants. The distance between δ nitrogen of His438 to ε oxygen of Glu325 and ε nitrogen of His438 to γ oxygen of Ser198 were highly altered in L307P mutant than the wild and other three variants throughout the simulation. Such disparity of distances between the catalytic residues may be due to the change in the protein conformation attributing to their differential catalytic activity. Our studies thus prove that the Indian BChE L307P mutant with negligible activity is possibly due to its structural instability when compared to other BChE variants.

Protein kinases are important regulators of many different cellular processes such as transcriptional control, cell cycle progression and differentiation, and have drawn much attention as potential drug targets. Leishmania mexicana mitogen-activated protein kinase 4 (LmxMPK4) is crucial for the survival of the parasite. As the crystal structure of the enzyme is not known, we have used bioinformatics techniques to model LmxMPK4 structure. The current study reveals conservation of all sequence and structural motifs of LmxMPK4. Study shows mitogen-activated protein kinases are highly conserved throughout different Leishmania species and significant divergence is observed towards mammalian mitogen-activated protein kinases. Additionally, using virtual docking methods, we have identified inhibitors for LmxMPK4. The sequence and structure analysis results were helpful in identifying the ligand binding sites and molecular function of the Leishmania specific mitogen-activated protein kinase.

Neurotransmitter transporter are found on the presynaptic neurons and on glial cells. The function of these transporters is termination of neurotransmission by the rapid removal of neurotransmitter molecules from the synaptic cleft. These transporters couple substrate transport to ion gradients of sodium and chloride. Structural studies often require the transporter to be removed from its physiological membrane, which can affect its structure or conformation. Crystal structures of the bacterial homolog LeuT (SLC6 family) were solved in three states of the transport cycle: occluded, outward and inward. The recent inward facing structure shows a conformation where the first helix (TM1A) did not seem to be compatible with a membrane environment. We carried out molecular dynamics simulations of LeuT in membrane and micelle environment to investigate the conformational behaviour of TM1A and combined the investigation with distance measurements using LRET. We used POPC as membrane lipids, and build the micelle systems with three different protein-detergent ratios (1:120, 1:140, or 1:160) using the detergent n-Octyl-β-D-Glucopyranoside (BOG) molecules. We observed a rigid body motion of the TM1A helix: it moves out of the hydrophobic core of the membrane. In contrary, TM1A was stable in its position in the micelle simulations. We confirmed this observation by distance measurements of solubilized LeuT in micelles and reconstituted POPC liposomes. This study suggests that the polar part of TM1A helix would not protrude into the membrane core. To further characterize the conformation of the TM1A helix, we used SMD simulations and pulled this helix relative to the scaffold or the core domain. The free energy profile was also endorsed our findings in the position of TM1A. These study indicates that changes in the environment can affect the equilibrium conformation of LeuT.

Background Glutamate transporters are integral membrane proteins that catalyze the concentrative uptake of glutamate from the synapse by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function; they also are implicated in stroke, epilepsy and neurodegenerative diseases. The crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii, is available at various conformations providing a structural framework for the determination of substrate and inhibitor binding to the transporter. In this study we aim to measure structural changes upon transport using lanthanide resonance energy transfer (LRET).

Structure-based virtual screening of NCI Diversity set II compounds was performed to indentify novel inhibitor scaffolds of trypanothione reductase (TR) fromLeishmania infantum. The top 50 ranked hits were clustered using the AuPoSOM tool. Majority of the top-ranked compounds were Tricyclic. Clustering of hits yielded four major clusters each comprising varying number of subclusters differing in their mode of binding and orientation in the active site. Moreover, for the first time, we report selected alkaloids and dibenzothiazepines as inhibitors ofLeishmania infantumTR. The mode of binding observed among the clusters also potentiates the probablein vitroinhibition kinetics and aids in defining key interaction which might contribute to the inhibition of enzymatic reduction of T[S] 2. The method provides scope for automation and integration into the virtual screening process employing docking softwares, for clustering the small molecule inhibitors based upon protein-ligand interactions.

Leishmaniasis is a parasitic infection caused by unicellular protozoan organism belonging to the family Trypanosomatidae. Among various forms of the disease, visceral leishmaniasis is the most lethal and caused by Leishmania infantum or Leishmania donovani. The redox metabolism of parasite requires a key enzyme, trypanothione reductase which is a validated drug target. In the past decade, it was observed that these protozoan parasites had developed resistance against many of available drugs. Importantly in the case of visceral leishmaniasis drug resistance is very high in the Indian subcontinent, a major endemic region of Leishmania donovani infection. In search for new drugs, we aim to identify potential natural product inhibitors of trypanothione reductase which can be further developed as anti-leishmanial drug. We have performed in silico virtual screening of a natural product data set of 800 diverse chemical entities. Leishmania infantum trypanothione reductase crystal structure (PDB ID: 2JK6) was used in the virtual screening process, docking studies to identify potential lead compounds. The compounds were sorted based upon their binding energy and the top 50 ranked protein-inhibitor complexes were clustered using AuPosSOM to ligand foot print the interactions. We report a few alkaloids and sterols for the first time, which could be potential trypanothione reductase inhibitors. The footprinting of protein-inhibitor interactions into clusters has also provided clues on various possible orientations that inhibitors can attain at the active site of Trypanothione reductase. Moreover, biological significance of the interactions has also been discussed.

Visceral leishmaniasis, most lethal form of Leishmaniasis, is caused by Leishmania infantum in the Old world. Current therapeutics for the disease is associated with a risk of high toxicity and development of drug resistant strains. Thiol-redox metabolism involving trypanothione and trypanothione reductase, key for survival of Leishmania, is a validated target for rational drug design. Recently published structure of trypanothione reductase (TryR) from L. infantum, in oxidized and reduced form along with Sb(III), provides vital clues on active site of the enzyme. In continuation with our attempts to identify potent inhibitors of TryR, we have modeled binding modes of selected tricyclic compounds and quinone derivatives, using AutoDock4. Here, we report a unique binding mode for quinone derivatives and 9-aminoacridine derivatives, at the FAD binding domain. A conserved hydrogen bonding pattern was observed in all these compounds with residues Thr335, Lys60, His461. With the fact that these residues aid in the orientation of FAD towards the active site forming the core of the FAD binding domain, designing selective and potent compounds that could replace FAD in vivo during the synthesis of Trypanothione reductase can be deployed as an effective strategy in designing new drugs towards Leishmaniasis. We also report the binding of Phenothiazine and 9-aminoacridine derivatives at the Z site of the protein. The biological significance and possible mode of inhibition by quinone derivatives, which binds to FAD binding domain, along with other compounds are discussed.

Synaptic transmission is regulated by the coordinated action of neurotransmitter release and reuptake. Specialized secondary-active neurotransmitter transporter proteins mediate the presynaptic reuptake of the respective neurotransmitters. Thereby, transporters re-accumulate the neurotransmitter in an economical fashion from the synaptic cleft into the presynaptic specialization by using the pre-established sodium gradient as a driving force. Two major classes can be distinguished: The During the last decade, crystal structures of archeal homologues of mammalian transporters have been published in various states. These structures serve as starting templates to study the structure function relationship in the mammalian counterparts. Hence, we combine molecular dynamics simulations and homology modeling with Lanthanide resonance energy transfer (LRET) measurements to explore substrate translocation in bacterial and mammalian transporters in a triangulated manner. We use the leucine/alanine transporter from Aquifex aeolicus (LeuTAa), the aspartate transporter from Pyrococcus horikoshi (GltPh) and the mammalian excitatory amino acid transporter 3 (EAAT3) and complement our key findings using biochemical studies. Using this multi-facetted approach allows us to ascertain the molecular movements of neurotransmitter transporters and their bacterial homologues and compare our results to the results obtained in crystallization experiments.

Background EAAT3 (excitatory amino acid transporter 3) mediates the regulation of synaptic transmission by reuptake of glutamate in the synaptic cleft. It is distributed in neuronal membranes and is selectively enriched in the neurons of the hippocampus, cerebellum and the basal ganglia. It belongs to the family of soluble carrier family 1 member 1 (SLC1A1) and is expressed in kidney, a wide variety of epithelial tissues, brain and eyes.