Growing tool-kit of photosensitizers for clinical and non-clinical applications.

Photosensitizers are photosensitive molecules utilized in clinical and non-clinical applications by taking advantage of light-mediated reactive oxygen generation, which triggers local and systemic cellular toxicity. Photosensitizers are used for diverse biological applications such as spatio-temporal inactivation of a protein in a living system by chromophore-assisted light inactivation, localized cell photoablation, photodynamic and immuno-photodynamic therapy, and correlative light-electron microscopy imaging. Substantial efforts have been made to develop several genetically encoded, chemically synthesized, and nanotechnologically driven photosensitizers for successful implementation in redox biology applications. Genetically encoded photosensitizers (GEPS) or reactive oxygen species (ROS) generating proteins have the advantage of using them in the living system since they can be manipulated by genetic engineering with a variety of target-specific genes for the precise spatio-temporal control of ROS generation. The GEPS variety is limited but is expanding with a variety of newly emerging GEPS proteins. Apart from GEPS, a large variety of chemically- and nanotechnologically-empowered photosensitizers have been developed with a major focus on photodynamic therapy-based cancer treatment alone or in combination with pre-existing treatment methods. Recently, immuno-photodynamic therapy has emerged as an effective cancer treatment method using smartly designed photosensitizers to initiate and engage the patient's immune system so as to empower the photosensitizing effect. In this review, we have discussed various types of photosensitizers, their clinical and non-clinical applications, and implementation toward intelligent efficacy, ROS efficiency, and target specificity in biological systems.

Structure of fish Toll-like receptors (TLR) and NOD-like receptors (NLR).

Innate immunity driven by pattern recognition receptor (PRR) protects the host from invading pathogens. Aquatic animals like fish where the adaptive immunity is poorly developed majorly rely on their innate immunity modulated by PRRs like toll-like receptors (TLR) and NOD-like receptors (NLR). However, current development to improve the fish immunity via TLR/NLR signaling is affected by a poor understanding of its mechanistic and structural features. This review discusses the structure of fish TLRs/NLRs and its interaction with pathogen associated molecular patterns (PAMPs) and downstream signaling molecules. Over the past one decade, significant progress has been done in studying the structure of TLRs/NLRs in higher eukaryotes; however, structural studies on fish innate immune receptors are undermined. Several novel TLR genes are identified in fish that are absent in higher eukaryotes, but the function is still poorly understood. Unlike the fundamental progress achieved in developing antagonist/agonist to modulate human innate immunity, analogous studies in fish are nearly lacking due to structural inadequacy. This underlies the importance of exploring the structural and mechanistic details of fish TLRs/NLRs at an atomic and molecular level. This review outlined the mechanistic and structural basis of fish TLR and NLR activation.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-ß Generates Toxic Hetero-oligomers.

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and ß-amyloid (Aß) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aß(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aß(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aß(M1-42) heteromers comprised of unstructured Aß(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aß(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aß(M1-42) is electrostatically driven and induces unfolding of Aß(M1-42). Neurotoxicity profiling of Aß(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aß(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation.

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

The ATP Transporter VNUT Mediates Induction of Dectin-1-Triggered Candida Nociception.

Candida albicans infection can cause skin, vulvar, or oral pain. Despite the obvious algesic activity of C. albicans, the molecular mechanisms of fungal nociception remain largely unknown. Here we show that the C. albicans-specific signaling pathway led to severe mechanical allodynia. We discovered that C. albicans-derived ß-glucan stimulated nociceptors depending on Dectin-1, and two pathways in inflammatory pain. The major pathway operates via the Dectin-1-mediated ATP-P2X3/P2X2/3 axis through intercellular relationships between keratinocytes and primary sensory neurons, which depends on the ATP transporter vesicular nucleotide transporter (VNUT). The other pathway operates via the Dectin-1-mediated PLC-TRPV1/TRPA1 axis in primary sensory neurons. Intriguingly, C. albicans-derived ß-glucan has the ability to enhance histamine-independent pruritus, and VNUT inhibitor clodronate can be used to treat unpleasant feelings induced by ß-glucan. Collectively, this is the first report to indicate that Dectin-1 and VNUT mediated innate sensory mechanisms that detect fungal infection.

Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs.

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Nociceptors Boost the Resolution of Fungal Osteoinflammation via the TRP Channel-CGRP-Jdp2 Axis.

Candida albicans can enter skeletal tissue through a skin wound in an immunocompromised host or by contamination during orthopedic surgery. Such Candida osteomyelitis is accompanied by severe pain and bone destruction. It is established that nociceptor innervation occurs in skin and bone, but the mechanisms of nociceptive modulation in fungal inflammation remain unclear. In this study, we show that C. albicans stimulates Nav1.8-positive nociceptors via the ß-glucan receptor Dectin-1 to induce calcitonin gene-related peptide (CGRP). This induction of CGRP is independent of Bcl-10 or Malt-1 but dependent on transient receptor potential cation channel subfamily V member 1 (TRPV1)/transient receptor potential cation channel subfamily A member 1 (TRPA1) ion channels. Hindpaw ß-glucan injection after Nav1.8-positive nociceptor ablation or in TRPV1/TRPA1 deficiency showed dramatically increased osteoinflammation accompanied by impaired CGRP production. Strikingly, CGRP suppressed ß-glucan-induced inflammation and osteoclast multinucleation via direct suppression of nuclear factor-κB (NF-κB) p65 by the transcriptional repressor Jdp2 and inhibition of actin polymerization, respectively. These findings clearly suggest a role for Dectin-1-mediated sensocrine pathways in the resolution of fungal osteoinflammation.

Conformational states of HAMP domains interacting with sensory rhodopsin membrane systems: an integrated all-atom and coarse-grained molecular dynamics simulation approach.

Understanding the downstream signaling mechanism of sensory rhodopsin and its cognate transducer complex (srII-htrII) has long been a challenge in the field of photoreceptor research. Here, an integration of all-atom and coarse-grained (CG) molecular dynamics (MD) simulations in different srII-htrII complex states is carried out. It is shown that the cytoplasmic four-helix HAMP dimer gives rise to a gear-box model interaction with discrete hydrophobic packing in Natronomonas pharaonis (Np). Structural analysis in all-atom and CG-MD reveals a stable conformational state in the physiological environment (323 K and 1.15 M salt). Comparative analysis in the ground and intermediate state conformations reveals substantial inter-HAMP interactions in the intermediate state with uniform clockwise (+10° to +30°) and counterclockwise (-20° to -40°) rotations in the α1 helix and the α2 helix of the monomer, respectively. Low temperature and low salt environments (283 K and 0.15 M) significantly affect srII-htrII binding affinity in both states with unusual helix bending. The distinguished control cable, knob-into-holes packing and piston-like movements in HAMP helices are found in the intermediate state complex. The N-terminal htrII (159 residues) coupled with srII yields a binding energy (ΔGbind) of -309.22, -436.53 and -331.11 kJ mol-1 in the MM/PBSA calculation for the NphtrII homodimer, the NpsrII-htrII ground state conformation and the NpsrII-htrII intermediate state conformation, respectively. Only the HAMP1 domain shows a very low ΔGbind value (-21.03 kJ mol-1) for the ground state in comparison to that for the intermediate state (-54.68 kJ mol-1). The structural analysis highlights the key residues that include Y199srII, T189srII, E43htrII, T86htrII, M100htrII, E116htrII, E126htrII and S130htrII for complex stabilization and signal transduction.

Membrane Mediated Antimicrobial and Antitumor Activity of Cathelicidin 6: Structural Insights from Molecular Dynamics Simulation on Multi-Microsecond Scale.

The cathelicidin derived bovine antimicrobial peptide BMAP27 exhibits an effective microbicidal activity and moderate cytotoxicity towards erythrocytes. Irrespective of its therapeutic and multidimensional potentiality, the structural studies are still elusive. Moreover, the mechanism of BMAP27 mediated pore formation in heterogeneous lipid membrane systems is poorly explored. Here, we studied the effect of BMAP27 in model cell-membrane systems such as zwitterionic, anionic, thymocytes-like (TLM) and leukemia-like membranes (LLM) by performing molecular dynamics (MD) simulation longer than 100 µs. All-atom MD studies revealed a stable helical conformation in the presence of anionic lipids, however, significant loss of helicity was identified in TLM and zwitterionic systems. A peptide tilt (~45˚) and central kink (at residue F10) was found in anionic and LLM models, respectively, with an average membrane penetration of < 0.5 nm. Coarse-grained (CG) MD analysis on a multi-µs scale shed light on the membrane-dependent peptide and lipid organization. Stable micelle and end-to-end like oligomers were formed in zwitterionic and TLM models, respectively. In contrast, unstable oligomer formation and monomeric BMAP27 penetration were observed in anionic and LLM systems with selective anionic lipid aggregation (in LLM). Peptide penetration up to ~1.5 nm was observed in CG-MD systems with the BMAP27 C-terminal oriented towards the bilayer core. Structural inspection suggested membrane penetration by micelle/end-to-end like peptide oligomers (carpet-model like) in the zwitterionic/TLM systems, and transmembrane-mode (toroidal-pore like) in the anionic/LLM systems, respectively. Structural insights and energetic interpretation in BMAP27 mutant highlighted the role of F10 and hydrophobic residues in mediating a membrane-specific peptide interaction. Free energy profiling showed a favorable (-4.58 kcal mol-1 for LLM) and unfavorable (+0.17 kcal mol-1 for TLM) peptide insertion in anionic and neutral systems, respectively. This determination can be exploited to regulate cell-specific BMAP27 cytotoxicity for the development of potential drugs and antibiotics.

Activity of a novel sulfonamide compound 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide against Leishmania donovani.

New treatments for visceral leishmaniasis, caused by Leishmania donovani, are needed to overcome sustained toxicity, cost, and drug resistance. The aim of this study was to evaluate the therapeutic effects of 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (2NB) against promastigote and amastigote forms of L. donovani and examine its effect in combination with amphotericin B (AmB) against AmB-resistant clinical isolates. Effects were assessed against extracellular promastigotes in vitro and intracellular amastigotes in L. donovani-infected macrophages. Levels of inducible nitric oxide and Th1 and Th2 cytokines were measured in infected 2NB-treated macrophages, and levels of reactive oxygen species and NO were measured in 2NB-treated macrophages. 2NB was active against promastigotes and intracellular amastigotes with 50% inhibitory concentration values of 38.5±1.5 µg/mL and 86.4±2.4 µg/mL, respectively. 2NB was not toxic to macrophages. Parasite titer was reduced by >85% in infected versus uninfected macrophages at a 2NB concentration of 120 µg/mL. The parasiticidal activity was associated with increased levels of Th1 cytokines, NO, and reactive oxygen species. Finally, 2NB increased the efficacy of AmB against AmB-resistant L. donovani. These results demonstrate 2NB to be an antileishmanial agent, opening up a new avenue for the development of alternative chemotherapies against visceral leishmaniasis.

Structural and functional insights into CARDs of zebrafish (Danio rerio) NOD1 and NOD2, and their interaction with adaptor protein RIP2.

Nucleotide-binding and oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern-recognition receptors (PRRs) composed of an N-terminal caspase activation and recruitment domain (CARD), a central NACHT domain and C-terminal leucine-rich repeats (LRRs). They play a vital role in innate immune signaling by activating the NF-κB pathway via recognition of peptidoglycans by LRRs, and ATP-dependent self-oligomerization of NACHT followed by downstream signaling. After oligomerization, CARD/s play a crucial role in activating downstream signaling via the adaptor molecule, RIP2. Due to the inadequacy of experimental 3D structures of CARD/s of NOD2 and RIP2, and results from differential experimental setups, the RIP2-mediated CARD-CARD interaction has remained as a contradictory statement. We employed a combinatorial approach involving protein modeling, docking, molecular dynamics simulation, and binding free energy calculation to illuminate the molecular mechanism that shows the possible involvement of either the acidic or basic patch of zebrafish NOD1/2-CARD/a and RIP2-CARD in CARD-CARD interaction. Herein, we have hypothesized 'type-I' mode of CARD-CARD interaction in NOD1 and NOD2, where NOD1/2-CARD/a involve their acidic surfaces to interact with RIP2. Asp37 and Glu51 (of NOD1) and Arg477, Arg521 and Arg529 (of RIP2) were identified to be crucial for NOD1-RIP2 interaction. However, in NOD2-RIP2, Asp32 (of NOD2) and Arg477 and Arg521 (of RIP2) were anticipated to be significant for downstream signaling. Furthermore, we found that strong electrostatic contacts and salt bridges are crucial for protein-protein interactions. Altogether, our study has provided novel insights into the RIP2-mediated CARD-CARD interaction in zebrafish NOD1 and NOD2, which will be helpful to understand the molecular basis of the NOD1/2 signaling mechanism.

Structural models of zebrafish (Danio rerio) NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Molecular recognition of avirulence protein (avrxa5) by eukaryotic transcription factor xa5 of rice (Oryza sativa L.): insights from molecular dynamics simulations.

The avirulence gene avrxa5 of bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) recognized by the resistant rice lines having corresponding resistance (xa5) gene in a gene-for-gene manner. We used a combinatorial approach involving protein-protein docking, molecular dynamics (MD) simulations and binding free energy calculations to gain novel insights into the gene-for-gene mechanism that governs the direct interaction of R-Avr protein. From the best three binding poses predicted by molecular docking, MD simulations were performed to explore the dynamic binding mechanism of xa5 and avrxa5. Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) techniques were employed to calculate the binding free energy and to uncover the thriving force behind the molecular recognition of avrxa5 by eukaryotic transcription factor xa5. Binding free energy analysis revealed van der Waals term as the most constructive component that favors the xa5 and avrxa5 interaction. In addition, hydrogen bonds (H-bonds) and essential electrostatic interactions analysis highlighted amino acid residues Lys54/Asp870, Lys56/Ala868, Lys56/Ala866, Lys56/Glu871, Ile59/His862, Gly61/Phe858, His62/Arg841, His62/Leu856, Ser101/Ala872 and Ser105/Asp870 plays pivotal role for the energetically stability of the R-Avr complex. Insights gained from the present study are expected to unveil the molecular mechanisms that define the transcriptional activator mediated transcriptome modification in host plants.

Understanding the distinguishable structural and functional features in zebrafish TLR3 and TLR22, and their binding modes with fish dsRNA viruses: an exploratory structural model analysis.

Viral infections are one of the major challenges in aquaculture production, and considered as the potential threat for fish farming. Toll-like receptor (TLR) 3 and TLR22 are highly specialized innate immune receptors that recognize double-stranded (ds)-RNA of viruses resulting in the induction of innate immunity. The existence of TLR3 and TLR22 only in aquatic animals indicates their distinctive characteristics in viral infection; however, the studies in exploring their structural features and dsRNA binding mechanism are still elusive. Here, we studied the structural and functional differentiations of TLR3 and TLR22 in zebrafish by employing comparative modeling and molecular dynamics simulation. Comparative structural analysis revealed a distinct spatial arrangement of TLR22 ectodomain with a flattened horseshoe-shape conformation as compared to other TLRs. Essential dynamics studies showed that unlike TLR3, TLR22 possessed a prominent motion, elasticity and twisting at both terminus separated by a distance equivalent to the length of a short-sized dsRNA. Interaction analysis of polyinosinic:polycytidylic acid (poly I:C) and dsRNA depicted leucine-rich-repeats (LRR)2-3 and LRR18-19 (in TLR3) and LRRNT-LRR3 and LRR22-24 (in TLR22) as the potential binding sites. The short-sized dsRNA binds tightly across its full-length with TLR22-monomer, and suggested that TLR22 dimer may sense long-sized dsRNA. Binding energy (BE) calculation using MM/PBSA method from the TLR3- and TLR22-ligand complexes revealed an adequate binding affinity between TLR22-monomer and dsRNA as like as TLR3-dimer-dsRNA complex. Mutagenesis and BE computation of key residues suggested their involvement in dsRNA recognition. These findings can be helpful for therapeutic applications against viral diseases in fish.

Structural and functional investigation of zebrafish (Danio rerio) NOD1 leucine rich repeat domain and its interaction with iE-DAP.

Nucleotide binding and oligomerization domain 1 (NOD1), a cytoplasmic pattern recognition receptor (PRR) and is a key component for modulating innate immunity and signaling. It is highly specific to γ-D-Glu-mDAP (iE-DAP), a cell wall component of Gram-negative and few Gram-positive bacteria. In the absence of the experimental structure of NOD1 leucine rich repeat (NOD1-LRR) domain, the NOD signaling cascade mediated through NOD1 and iE-DAP interaction is poorly understood. Herein, we modeled 3D structure of zebrafish NOD1-LRR (zNOD1-LRR) through a protein-threading approach and structural integrity of the model was assessed using molecular dynamics simulations. Molecular interaction analysis of iE-DAP and zNOD1-LRR, their complex stability and binding free energy studies were conducted to anticipate the ligand binding residues in zNOD1. Our study revealed that His775, Lys777, Asp803, Gly805, Trp807, Asn831, Ser833, Ile859 and Trp861 situated in the ß-sheet region of zNOD1-LRR could be involved in iE-DAP recognition, which correlates the earlier findings in human. Comparison of binding free energies of native and mutant zNOD1-iE-DAP complexes delineated His775, Lys777, Asp803, Ser833 and Ile859 as the pivotal residues for energetic stability of NOD1 and iE-DAP interaction. This study provides the first comprehensive description of biophysical and biochemical parameters responsible for NOD1 and iE-DAP interaction in zebrafish, which is expected to shed more light on NOD1 signaling and therapeutic applications in other organisms.

LRRsearch: An asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors.

The leucine-rich repeat (LRR) motifs of the nucleotide-binding oligomerization domain like receptors (NLRs) play key roles in recognizing and binding various pathogen associated molecular patterns (PAMPs) resulting in the activation of downstream signaling and innate immunity. Therefore, identification of LRR motifs is very important to study ligand-receptor interaction. To date, available resources pose restrictions including both false negative and false positive prediction of LRR motifs from the primary protein sequence as their algorithms are relied either only on sequence based comparison or alignment techniques or are over biased for a particular LRR containing protein family. Therefore, to minimize the error (≤5%) and to identify a maximum number of LRR motifs in the wide range of proteins, we have developed "LRRsearch" web-server using position specific scoring matrix (PSSM) of 11 residue LRR-HCS (highly conserved segment) which are frequently observed motifs in the most divergent classes of LRR containing proteins. A data library of 421 proteins, distributed among five known NLR families has also been integrated with the "LRRsearch" for the rich user experience. The access to the "LRRsearch" program is freely available at http://www.lrrsearch.com/.

Exploration of the binding modes of buffalo PGRP1 receptor complexed with meso-diaminopimelic acid and lysine-type peptidoglycans by molecular dynamics simulation and free energy calculation.

The peptidoglycan recognition proteins (PGRPs) are the key components of innate-immunity, and are highly specific for the recognition of bacterial peptidoglycans (PGN). Among different mammalian PGRPs, the PGRP1 binds to murein PGN of Gram-positive bacteria (lysine-type) and also have bactericidal activity towards Gram-negative bacteria (diaminopimelic acid or Dap-type). Buffaloes are the major sources of milk and meat in Asian sub-continents and are highly exposed to bacterial infections. The PGRP activates the innate-immune signaling, but their studies has been confined to limited species due to lack of structural and functional information. So, to understand the structural constituents, 3D model of buffalo PGRP1 (bfPGRP1) was constructed and conformational and dynamics properties of bfPGRP1 was studied. The bfPGRP1 model highly resembled human and camel PGRP structure, and shared a highly flexible N-terminus and centrally placed L-shaped cleft. Docking simulation of muramyl-tripeptide, tetrapeptide, pentapeptide-Dap-(MTP-Dap, MTrP-Dap and MPP-Dap) and lysine-type (MTP-Lys, MTrP-Lys and MPP-Lys) in AutoDock 4.2 and ArgusLab 4.0.1 anticipated ß1, α2, α4, ß4, and loops connecting ß1-α2, α2-ß2, ß3-ß4 and α4-α5 as the key interacting domains. The bfPGRP1-ligand complex molecular dynamics simulation followed by free binding energy (BE) computation conceded BE values of -18.30, -35.53, -41.80, -25.03, -24.62 and -22.30 kJ mol(-1) for MTP-Dap, MTrP-Dap, MPP-Dap, MTP-Lys, MTrP-Lys and MPP-Lys, respectively. The groove-surface and key binding residues involved in PGN-Dap and Lys-type interaction intended by the molecular docking, and were also accompanied by significant BE values directed their importance in pharmacogenomics, and warrants further in vivo studies for drug targeting and immune signaling pathways exploration.

Structural and dynamic investigation of bovine folate receptor alpha (FOLR1), and role of ultra-high temperature processing on conformational and thermodynamic characteristics of FOLR1-folate complex.

The folate receptor alpha (FOLR1) present in milk has widely been studied to investigate the effects of pasteurization, ultra-high temperature (UHT) processing and fermentation on net folate concentration. However, the folate binding mechanism with FOLR1, and effect of temperature on FOLR1-folate complex is poorly explored till now in bovine milk which is a chief resource of folate. Despite of enormous importance of folic acid and the routine intake of bovine milk, folic acid deficiency diseases are common in human race. To understand the folate deficiency in milk after processing, in absence of experimental structure, 3D model of bovine FOLR1 (bvFOLR1) was built followed by 40ns molecular dynamics (MD) simulation. The folate and its derivatives binding sites in bvFOLR1 were anticipated by molecular docking using AutoDock 4.2. Essential MD studies suggested the presence of a longer signal peptide (22 residues) and a short propeptide (7 residues) at the C-terminus that may cleaved during post-translational modification. MD analysis of bvFOLR1-folate complex at 298, 323, 353, 373 and 408K followed by binding energy (BE) calculation showed maximum binding affinity at ∼353K. However, at 373K and UHT (408K), the folate BE is significantly decreased with substantial conformational alteration. Heating at UHT followed by cooling within 298-408K range demoed no structural reformation with temperature reduction, and the folate was displaced from the active site. This study presented the disintegration of folate from bvFOLR1 during high temperature processing and revealed a lower folate concentration in UHT milk and dairy products.

Structural insights into the MDP binding and CARD-CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach.

Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)-a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2-leucine rich repeat (LRR) domain activates NF-κB signaling through a protein-protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD-CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD-CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2-LRR confer MDP recognition by hydrophobic and hydrogen bond (H-bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2-CARDa and the electronegative surface on zRIP2-CARD reinforced mostly by H-bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2-CARDa and Asp494 of zRIP2-CARD. Arg11 and Lys53 of zNOD2-CARDa and Ser498 and Glu508 of zRIP2-CARD are critical residues for CARD-CARD interaction and NOD2 signaling. The 2.7 Å H-bond between Lys104 of the linker and Glu508 of zRIP2-CARD suggests a possible role of the linker for shaping CARD-CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD-CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective.

Elucidation of novel structural scaffold in rohu TLR2 and its binding site analysis with peptidoglycan, lipoteichoic acid and zymosan ligands, and downstream MyD88 adaptor protein.

Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)-ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16-19, LRR12-14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)-protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, α B-helix, α C-helix, and CD loop in TLR2-TIR and BB loop, α B-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling.