Insights into the dynamic interactions at chemokine-receptor interfaces and mechanistic models of chemokine binding.

Chemokine receptors are the central signaling hubs of several processes such as cell migration, chemotaxis and cell positioning. In this graphical review, we provide an overview of the structural and mechanistic principles governing chemokine recognition that are currently emerging. Structural models of chemokine-receptor co-complexes with endogenous chemokines, viral chemokines and therapeutics have been resolved that highlight multiple interaction sites, termed as CRS1, CRS1.5 etc. The first site of interaction has been shown to be the N-terminal domain of the receptors (CRS1 site). A large structural flexibility of the N-terminal domain has been reported that was supported by both experimental and simulation studies. Upon chemokine binding, the N-terminal domain appears to show constricted dynamics and opens up to interact with the chemokine via a large interface. The subsequent sites such as CRS1.5 and CRS2 sites have been structurally well resolved although differences arise such as the localization of the N-terminus of the ligand to a major or minor pocket of the orthosteric binding site. Several computational studies have highlighted the dynamic protein-protein interface at the CRS1 site that seemingly appears to resolve the differences in NMR and mutagenesis studies. Interestingly, the differential dynamics at the CRS1 site suggests a mixed model of binding with complex signatures of both conformational selection and induced fit models. Integrative experimental and computational approaches could help unravel the structural basis of promiscuity and specificity in chemokine-receptor binding and open up new avenues of therapeutic design.

Allosteric modulation of the chemokine receptor-chemokine CXCR4-CXCL12 complex by tyrosine sulfation.

The chemokine receptor CXCR4 and its cognate ligand CXCL12 mediate pathways that lead to cell migration and chemotaxis. Although the structural details of related receptor-ligand complexes have been resolved, the roles of the N-terminal domain of the receptor and post-translational sulfation that are determinants of ligand selectivity and affinity remain unclear. Here, we analyze the structural dynamics induced by receptor sulfation by combining molecular dynamics, docking and network analysis. The sulfotyrosine residues, 7YsN-term, 12YsN-term and 21YsN-term allow the N-terminal domain of the apo-sulfated receptor to adopt an "open" conformation that appears to facilitate ligand binding. The overall topology of the CXCR4-CXCL12 complex is independent of the sulfation state, but an extensive network of protein-protein interactions characterizes the sulfated receptor, in line with its increased ligand affinity. The altered interactions of sulfotyrosine residues, such as 21YsN-term-47RCXCL12 replacing the 21YN-term-13FCXCL12 interaction, propagate via allosteric pathways towards the receptor lumen. In particular, our results suggest that the experimentally-reported receptor-ligand interactions 262D6.58-8RCXCL12 and 277E7.28-12RCXCL12 could be dependent on the sulfation state of the receptor and need to be carefully analyzed. Our work is an important step in understanding chemokine-receptor interactions and how post-translational modifications could modulate receptor-ligand complexes.

Role of Phosphorylation and Hyperphosphorylation of Tau in Its Interaction with ßα Dimeric Tubulin Studied from a Bioinformatics Perspective.

BACKGROUND: Tau is a disordered Microtubule Associated Protein (MAP) which prefers to bind and stabilize microtubules. Phosphorylation of tau in particular enhances tautubulin interaction which otherwise detaches from tubulin during hyperphosphorylation. The reason behind their destabilization, detachment and the role of ß subunit (from microtubule) and the projection domain (Tau) in microtubule stability remains elusive till date. Thus, a complete 3D structural investigation of tau protein is much needed to address these queries as the existing crystal structures are in fragments and quite limited. METHODS: In this study, the modelled human tau protein was subjected to phosphorylation and hyperphosphorylation which were later considered for docking with micro-tubules (ßα subunits-inter dimer) and vinblastine. RESULTS: Phosphorylated tau protein interacts with both α- and ß subunits. But stronger bonding was with α- compared to ß subunits. Regarding ß subunit, proline rich loop and projection domain actively participated in tau binding. Interestingly, hyperphosphorylation of tau increases MAP domain flexibility which ultimately results in tau detachment, the main reason behind tangle formation in Alzheimer's disease. CONCLUSION: This study being the first of its kind emphasizes the role of projection domain and proline rich region of ß-subunit in stabilizing the tau-tubulin interaction and also the effect of hyperphosphorylation in protein-protein and protein-drug binding.

Molecular docking analysis of hyperphosphorylated tau protein with compounds derived from Bacopa monnieri and Withania somnifera.

Tau protein, the major player in Alzheimer's disease forms neurofibrillary tangles in elderly people. Bramhi (Baccopa Monniera) is often used as an ayurvedic treatment for Alzheimer's disease. Therefore it is of interest to study the interaction of compounds derived from Baccopa with the Tau protein involved in tangle formation. We show that compounds such as bacopaside II, bacopaside XII, and nicotine showed optimal binding features with the R2 repeat domain of hyperphosphorylated tau protein for further consideration in the context of Alzheimer's disease (AD).

Insights into the flexibility of the T3 loop and GTPase activating protein (GAP) domain of dimeric α and ß tubulins from a molecular dynamics perspective.

Tubulin protein is the fundamental unit of microtubules, and comprises of α and ß subunits arranged in an alternate manner forming protofilaments. These longitudinal protofilaments are made up of intra- (α-ß) and inter-dimer (ß-α) interactions. Literature review confirms that GTP hydrolysis results in considerable structural rearrangement within GTP binding site of ß-α dimer interface after the release of γ phosphate. In addition to this, the intra-dimer interface exhibits structural rigidity which needs further investigation. In this study, we explored the reasons for the flexibility and the rigidity of the ß-α dimer and the α-ß dimer respectively through molecular simulation and Anisotropic Normal Mode based analysis. As per the sequence alignment report, two glycine residues (Gly96 and Gly98) were observed in the T3 loop of the ß subunit which get substituted by Asp98 and Ala100 in the T3 loop of the α subunit. The higher mobility of glycine residues contributes to the flexibility of the T3 loop of inter-dimer when they come in direct contact with the GTPase Activating Protein (GAP) domain of the subunit. This was confirmed through RMSD, RMSF and Radius of Gyration based studies. Conversely, the intra-dimer exhibited a lower mobility in the absence of glycine residues. As per ANM based analysis, positive domain correlations were observed between T3 loop and GAP domain of intra- and inter- dimeric contact regions. However, these correlation motions were higher in the intra-dimer as compared to the inter-dimer interface. Thus on the basis of our findings, we hypothesize that the higher flexibility of T3 loop and the GAP domain of the inter-dimer is required for structural rearrangement and protofilament stability during hydrolysis. Furthermore, the slightly rigid nature of the T3 loop and the GAP domain of the intra-dimer assists in enhancing the monomer-monomer interaction through the higher positive domain correlation.

Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C.

Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents--octyl-beta-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in V(max) (35%) without a significant change in K(m). Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation.

Comparative study of modified quantitative buffy coat and two rapid tests in comparison with peripheral blood smear in malaria diagnosis in mumbai, India.

In order to identify a quick and reliable technique for accurate diagnosis of malaria, study of the efficiency of the tests such as Parahit total (HRPII & aldolase Ag), Advantage mal card (parasite specific LDH), and modified QBC was done in comparison with conventional blood smear microscopy. One hundred patients infected with P. vivax and 101 infected with P. falciparum were included in this study. The sensitivity of Parahit total, Advantage mal card, and modified QBC for P. falciparum detection was 70.3, 95%, and 98%, and specificity was 98%, 98%, and 96%, respectively. The sensitivity of Parahit total, Advantage mal card, and modified QBC for P. vivax detection was 73%, 97.0%, and 98%, respectively, and specificity of all the tests was 98%. On day 15, in falciparum arm, Advantage mal card and Parahit total showed 8 (7.92%) and 59 (58.41%) false positives. On day 15, in vivax arm, Parahit total revealed 52% false positives. The study indicated that modified QBC could be only used where appropriate facilities are available. Advantage mal card was a better follow-up tool than Parahit total.

A preliminary comparative report of quantitative buffy coat and modified quantitative buffy coat with peripheral blood smear in malaria diagnosis.

The quantitative buffy coat (QBC) technique is a method of diagnosing malarial parasites based on micro-centrifugation, fluorescence, and density gradient of infected red blood cells. The aim of the present study was to modify the QBC technique in order to reduce the cost per test of malaria diagnosis. This was achieved by introducing some modifications to routine QBC wherein REMI centrifuge (cost Rs 19000/-) and ultra-violet microscope (Rs 115000) were used instead of parafuge (Rs 108000) and paralens (Rs 293625/-). With the above modification, the cost per test for laboratories dealing with high patient load was reduced by 13%, whereas for smaller laboratories with low patient load, the cost per test was reduced by 48%. This is a significant difference in cost. The results of the modified QBC method were compared with the current diagnostic methods: peripheral blood smear (PBS) and routine QBC. Blood samples collected from 96 patients were subjected to the above tests. Considering PBS as the gold standard, routine QBC showed 91% sensitivity and 96% specificity for Plasmodium vivax- and 91% sensitivity and 94% specificity for Plasmodium falciparum-infected patients. It was seen that the modified QBC technique had 91% sensitivity and 98% specificity for P. vivax and 91% sensitivity and 96% specificity for P. falciparum. It was concluded that modification of the QBC technique renders it cheaper without compromising the specificity and sensitivity of the method.

Does the growth temperature of a prokaryote influence the purine content of its mRNAs?

The formation and breaking of hydrogen bonds between nucleic acid bases are dependent on temperature. The high G+C content of organisms was surmised to be an adaptation for high temperature survival because of the thermal stability of G:C pairs. However, a survey of genomic GC% and optimum growth temperature (OGT) of several prokaryotes revoked any direct relation between them. Significantly high purine (R=A or G) content in mRNAs is also seen as a selective response for survival among thermophiles. Nevertheless, the biological relevance of thermophiles loading their unstable mRNAs with excess purines (purine-loading or R-loading) is not persuasive. Here, we analysed the mRNA sequences from the genomes of 168 prokaryotes (as obtained from NCBI Genome database) with their OGTs ranging from -5 °C to 100 °C to verify the relation between R-loading and OGT. Our analysis fails to demonstrate any correlation between R-loading of the mRNA pool and OGT of a prokaryote. The percentage of purine-loaded mRNAs in prokaryotes is found to be in a rough negative correlation with the genomic GC% (r(2)=0.655, slope=-1.478, P<000.1). We conclude that genomic GC% and bias against certain combinations of nucleotides drive the mRNA-synonymous (sense) strands of DNA towards variations in R-loading.

Screening of natural phenolic compounds for potential to inhibit bacterial cell division protein FtsZ.

FtsZ plays an important role in bacterial cell division by polymerizing to form the Z ring at the site of cytokinesis. Phytochemicals are known to disrupt bacterial cell division through inhibition of FtsZ assembly. In the present study phytochemicals like eugenol, trans-cinnamic acid, 4-formyl cinnamic acid, naringenin and caffeic acid were were tested for their potential to inhibit cell division. Effect of these antimicrobial compounds on the growth of E. coli was determined and the inhibition of FtsZ assembly in vitro was investigated. The present study revealed trans-cinnamic acid as the most potent inhibitor of FtsZ assembly.

Berberine targets assembly of Escherichia coli cell division protein FtsZ.

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.

Decolorisation of synthetic dyes and textile wastewater using Polyporus rubidus.

Effluent from textile industries were treated with enzyme from white rot fungi isolated from outskirts of Mumbai and identified as Polyporus rubidus in our laboratory. Decolorisation of 4 Reactive dyes commonly found in the effluents such as Reactive bue, Reactive orange, Ramazol black and Congo red was examined by treatment with enzyme from Polyporus rubidus. Treatment of effluent was done in a laboratory scale bioreactor constructed with laccase immobilized Na-alginate beads. Greater than 80% of dyes were degraded within 5 days under stationary incubation conditions. The enzyme had a maxmimum activity of 17.1U after 3 days and was found to be secreted extracellularly by Polyporus rubidus. In this study the Polyporus rubidus has been reported for the first time to have laccase activity offering a promising possibility to develop an easy and cost effective method for degradation of dangerous dyes.

Inhibition of bacterial cell division protein FtsZ by cinnamaldehyde.

Cinnamaldehyde is a natural product from spices that inhibits cell separation in Bacillus cereus. Cell division is regulated by FtsZ, a prokaryotic homolog of tubulin. FtsZ assembles into the Z-ring at the site of cell division. Here, we report the effect of cinnamaldehyde on FtsZ and hence on the cell division apparatus. Cinnamaldehyde decreases the in vitro assembly reaction and bundling of FtsZ. It is found that cinnamaldehyde perturbs the Z-ring morphology in vivo and reduces the frequency of the Z ring per unit cell length of Escherichia coli. In addition, GTP dependent FtsZ polymerization is inhibited by cinnamaldehyde. Cinnamaldehyde inhibits the rate of GTP hydrolysis and binds FtsZ with an affinity constant of 1.0+/-0.2 microM(-1). Isothermal titration calorimetry reveals that binding of cinnamaldehyde to FtsZ is driven by favorable enthalpic interactions. Further, we map the cinnamaldehyde binding region of FtsZ, using the saturation transfer difference-nuclear magnetic resonance and an in silico docking model. Both predict the cinnamaldehyde binding pocket at the C terminal region involving the T7 loop of FtsZ. Our results show that cinnamaldehyde binds FtsZ, perturbs the cytokinetic Z-ring formation and inhibits its assembly dynamics. This suggests that cinnamaldehyde, a small molecule of plant origin, is a potential lead compound that can be developed as an anti-FtsZ agent towards drug design.

Pyrene excimer fluorescence of yeast alcohol dehydrogenase: a sensitive probe to investigate ligand binding and unfolding pathway of the enzyme.

The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N-(1-pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM-YADH was found to be similar to that of the native YADH using far-UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM-YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM-YADH indicating that these agents induce conformational changes in the enzyme. Guanidinium hydrochloride (GdnHCl)-induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH.

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments.

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.