Amino Acid Residues For Binding Research Articles

Characterization and Evaluation of Key Sites in the Peptide Inhibitor of TAB1/p38α Interaction

A combination of computation techniques and peptide mutants have been used to determine the binding site and amino acid residues on the inhibitor peptide that are critical for binding to Mitogen-activated protein kinase 14 (p38α). In our previous research work, the functional peptide, named as PT5, target to p38α, was obtained based on the theoretical complex structure of p38α and [transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1] (TAB1). Based on the computer-guided ab initio modeling method, the inhibitor peptide PT5 and its mutants were modeled. Furthermore, the 3-D complex structures of PT5 or its mutants and p38α were constructed using molecular docking and dynamics simulation methods. The key residues in the peptide PT5 involved in binding interaction to p38α were predicted. According to the 3-D theoretical complex structure PT5/ p38α, the interaction binding mode between PT5 and p38α was analyzed using distance geometry technology. Mutants of the peptide PT5 was used to evaluate the bio-function when the critical residues were mutated. The mutant experimental results identified the key residues in PT5, i.e. Thr11 and Asp12 and determined the core sequence of PT5 binding to p38α. Based on the results, optimized peptides compounds could be developed for treating myocardial ischemia/reperfusion (I/R) injury in clinical.

Structural insights into the production of 3-hydroxypropionic acid by aldehyde dehydrogenase from Azospirillum brasilense

3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide. Aldehyde dehydrogenase (ALDH), an enzyme that catalyzes the reaction of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, determines 3-HP production rate during the conversion of glycerol to 3-HP. To elucidate molecular mechanism of 3-HP production, we determined the first crystal structure of a 3-HP producing ALDH, α-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH), in its apo-form and in complex with NAD+. Although showing an overall structure similar to other ALDHs, the AbKGSADH enzyme had an optimal substrate binding site for accepting 3-HPA as a substrate. Molecular docking simulation of 3-HPA into the AbKGSADH structure revealed that the residues Asn159, Gln160 and Arg163 stabilize the aldehyde- and the hydroxyl-groups of 3-HPA through hydrogen bonds, and several hydrophobic residues, such as Phe156, Val286, Ile288, and Phe450, provide the optimal size and shape for 3-HPA binding. We also compared AbKGSADH with other reported 3-HP producing ALDHs for the crucial amino acid residues for enzyme catalysis and substrate binding, which provides structural implications on how these enzymes utilize 3-HPA as a substrate.

Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins

A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (−89° to −60°, −59° to −30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

3D Homology Model of Sigma1 Receptor.

This chapter presents the three-dimensional (3D) model of the Sigma1 receptor protein as obtained from homology modeling techniques. We show the applicability of this structure to docking-based virtual screening and discuss combined in silico/in vitro mutagenesis studies performed to validate the structural features of the Sigma1 receptor model and to qualify/quantify the prominent role of specific amino acid residues in ligand binding. The validation of the virtual 3D Sigma1 receptor model and its reliable applicability to docking-based virtual screening is of significance for rational ligand design, even in light of the recently reported crystal structure for the Sigma1 receptor.

Exploring the Binding Affinity of Novel Syringic Acid Analogues and Critical Determinants of Selectivity as Potent Proteasome Inhibitors.

Syringic acid, a known plant phenolic compound and its analogues are known to possess high proteasome inhibitory activity. In the current work, we describe synthesis, characterization, DFT, docking of syringic acid (SA) and analogues (SAA1 and SAA2) and biological effects were studied. Syringic acid and its analogues were docked for the first time with the crystal structures of β5 proteasome of diverse eukaryotic organisms. Among all proteasomes, the humanoid proteasome showed the highest degree of docking conformation and low inhibition constant (Ki). SAA2 specifically displayed binding to the N-terminal Thr1 residue in the S1 pocket of Mus musculus β5 proteasome along with threonine, lysine and arginine; conventionally involved major amino acid residues in ligand binding. The geometrical properties (B3LYP/6- 31g (d, p)) and electrostatic potentials of molecules were computed using DFT calculations. A detailed molecular picture of the compounds and its interactions was obtained from NBO analysis. SA-analogues elucidated potent antioxidant activities and good antibacterial activity. In-vitro DNA binding studies revealed that all molecules had strong binding at the major groove of dsDNA. In the view of medical applicability, proteasome inhibition is an important therapeutic strategy for various types of cancers. Therefore, current discoveries may encourage the rational design and development of new chemical entities of syringic acid based chemotherapeutics.

The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Qo site of bacterial cytochrome bc1

The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn2+ binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1.

Signal Amplification Techniques By Effective Use of the Interfacial Region for Field Effect Transistor (FET) Based Biosensing

Highly sensitive method is greatly desirable for detecting protein biomarkers, which exist in most cases at very low concentration. Such performance would also be of utmost importance to the development of early detection test. In the past few years, we have demonstrated sensitive diagnosis of emerging protein diseases by using field effect transistor (FET) biosensor. Among the recent proposed biosensor devices, FET can offer a simple operation and applicable integration as well as miniaturization that are useful for large-scale production and low-cost application test. This promising FET biosensor principally detects the charged target molecules that specifically interact with probe molecule onto the FET gate surface within the Debye length. Therefore, we can construct strategy for signal amplification with the approaches from the probe molecules as well as from the charged target molecule. To date, we have successfully achieved several techniques to improve sensing signal for FET biosensor by applying small probe molecules[1-3] and by enhancing net charges of target molecules[4-6]. Application of small probe molecules Utilization of small probe molecule is beneficial to improve sensitivity of FET biosensor due to the molecular interaction taking place closer to the sensing surface, which leads to the increase of detectable region of charged target molecules within the Debye length. We have shown a good sensitivity of FET biosensor modified with antigen-binding fragment (Fab) instead of native antibody for detecting cancer biomarkers[1]. In addition, by taking the advantages of small probes, we succeeded sensitive detection for influenza virus-related protein and amyloid β protein (Aβ) using glycan[2] and Congo red[3], respectively. Enhancement of net charges of target molecules In this approach, the main idea is to intensify the charge of target molecules that cause an increase of charge density on the sensing surface. For the first case, we have designed a unique dual-ligand binding by adding the second ligand (metal ions) into the target of prion proteins, which clearly showed an enhancement of the sensitivity of FET biosensor[4]. Additionally, we performed a potent model of surfactant conjugated with target molecule that is useful for signal amplification using FET biosensor[5]. In both of these two examples, signal amplification was mainly due to additional charge by introducing other ions or molecules. Furthermore, we also established a straightforward strategy to enhance the charge of protein by direct modification of the concerned target molecule with chemical modification[6]. This technique proved a significant enhancement of signal amplification for the FET-based biosensing detection with high sensitivity. As described above, we proposed two strategies in the signal amplification for FET biosensors and demonstrated their usefulness. These strategies would be applicable to the detection of other proteins, with adjustment to a certain degree depending on binding affinity and amino acid residues of proteins.AcknowledgementsThis work is partly supported by the Center of Innovation Program, Building of Consortia for the Development of Human Resources in Science and Technology and Grant-in-Aid for Young Scientists (B) (16K17498), all from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.References S. Cheng, K. Hotani, S. Hideshima, S. Kuroiwa, T. Nakanishi, M. Hashimoto, Y. Mori, T. Osaka, Materials, 7, 2490–2500 (2014). S. Hideshima, H. Hinou, D. Ebihara, R. Sato, S. Kuroiwa, T. Nakanishi, S. Nishimura, T. Osaka, Analytical Chemistry, 85, 5641–5644 (2013). S. Hideshima, M. Kobayashi, T. Wada, S. Kuroiwa, T. Nakanishi, N. Sawamura, T. Asahi, T. Osaka, Chemical Communication, 50, 3476–3479 (2014). S. Wustoni, S. Hideshima, S. Kuroiwa, T. Nakanishi, M. Hashimoto, Y. Mori, T. Osaka, Biosensors and Bioelectronics, 67, 256-262 (2015). S. Hideshima, K. Fujita, Y. Harada, M. Tsuna, Y. Seto, S. Sekiguchi, S. Kuroiwa, T. Nakanishi T. Osaka, Sensing and Biosensing Research, 7, 90-94 (2016). S. Wustoni, S. Hideshima, S. Kuroiwa, T. Nakanishi, Y. Mori, T. Osaka, Sensors and Actuators B, 230, 374-379 (2016).

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters.

Novel in silico to in vitro schema: new antibiotic exploration approach

Docking prediction analysis is a computerized model which can aid in developing new potential bacterial targets for antibiotics. Trying to find a molecule with a high potential binding with its macromolecule (protein) is challenging. The bacterial cross talk (quorum sensing [QS]) mechanisms are bacterial regulation processes which allow bacteria to express pathogenic phenotypes such as biofilm formation. By targeting the QS components, a novel way of affecting bacterial behavior is achieved, possibly without creating antibiotic resistance. We conducted a combined analysis of a new synthetic drug with a docking in silico prediction applied on potential QS targets in Pseudomonas aeruginosa , and by doing so validated previous docking results of other QS inhibitors. We complemented the in silico analysis with an in vitro E. coli - QS reporting assay in order to confirm a new class of QS inhibitors. Our analysis was confirmed after conducting site-directed mutagenesis over the sequences of the potential binding amino acid residues of the suspected QS inhibitor target. Our analysis was supported by a wide meta-genomic microarray analysis which revalidated the QS inhibition schema. The QS inhibitor successfully exhibited a decrease in the pathogenic phenotypes of surface attached biofilm and swarming motility in P. aeruginosa .

Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.

Insight into determinants of substrate binding and transport in a multidrug efflux protein.

Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that ‘aromatic clamps’ formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such ‘clamps’ may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.

Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis.

Caerulomycin A (CRM A 1) belongs to a family of natural products containing a 2,2'-bipyridyl ring core structure and is currently under development as a potent novel immunosuppressive agent. Herein, we report the functional characterization, kinetic analysis, substrate specificity, and structure insights of an aminotransferase CrmG in 1 biosynthesis. The aminotransferase CrmG was confirmed to catalyze a key transamination reaction to convert an aldehyde group to an amino group in the 1 biosynthetic pathway, preferring l-glutamate and l-glutamine as the amino donor substrates. The crystal structures of CrmG in complex with the cofactor 5'-pyridoxal phosphate (PLP) or 5'-pyridoxamine phosphate (PMP) or the acceptor substrate were determined to adopt a canonical fold-type I of PLP-dependent enzymes with a unique small additional domain. The structure guided site-directed mutagenesis identified key amino acid residues for substrate binding and catalytic activities, thus providing insights into the transamination mechanism of CrmG.

Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation

We studied the kinetics and structural determinants of closed-state inactivation (CSI) in Kv4.2 channels, considering a multistep process and the possibility that both intra- and intersubunit dynamic binding (i.e., loss and restoration of physical contact) may occur between the S4-S5 linker, including the initial S5 segment (S4S5), and the S6 gate. We expressed Kv4.2 channels in Xenopus oocytes and measured the onset of low-voltage inactivation under two-electrode voltage clamp. Indicative of a transitory state, the onset kinetics were best described by a double-exponential function. To examine the involvement of individual S4S5 and S6 amino acid residues in dynamic binding, we studied S4S5 and S6 single alanine mutants and corresponding double mutants. Both transitory and steady-state inactivation were modified by these mutations, and we quantified the mutational effects based on apparent affinities for the respective inactivated states. Double-mutant cycle analyses revealed strong functional coupling of the S6 residues V404 and I412 to all tested S4S5 residues. To examine whether dynamic S4S5/S6 binding occurs within individual α-subunits or between neighboring α-subunits, we performed a double-mutant cycle analysis with Kv4.2 tandem-dimer constructs. The constructs carried either an S4S5/S6 double mutation in the first α-subunit and no mutation in the second (concatenated) α-subunit or an S4S5 point mutation in the first α-subunit and an S6 point mutation in the second α-subunit. Our results support the notion that CSI in Kv4.2 channels is a multistep process that involves dynamic binding both within individual α-subunits and between neighboring α-subunits.

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

BackgroundStarch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C.ResultsIn this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding.ConclusionsThe results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1598-6) contains supplementary material, which is available to authorized users.

<i>In Silico</i> Analysis of <i>Curcuma longa</i> Against PCAF Histon Asetiltransferase

Curcumin turmeric (C. longa) has been found capable of inhibiting p300/CBP associated factor (PCAF) histone asetyltransferase (HAT) as a cancer biomarker. However, the molecular mechanism of inhibition is unkown. This study is aimed to analyse the molecular inhibition mechanisms by in silico method. The analysis showed that curcumin’s Gibbs free energy (ΔG) was stable and capable of binding amino acid residues believed to be active site of PCAF HAT. Curcumin is predicted to be a PCAF HAT inhibitor, and further in vitro analysis is required.

The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence

Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD+-dependent ADH activity was diminished by 20–75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.

Structural characterization of Fis — A transcriptional regulator from pathogenic Pasteurella multocida essential for expression of virulence factors

Pasteurella multocida is responsible behind a variety of diseases in animals. The disease causing substance of this bacterium is a capsular polysaccharide. The expression of the gene that codes for the bacterial capsule is regulated by the protein Fis. Fis also regulates the expression of various different genes in P. multocida. So far there have been no previous reports that depict the characterization of Fis from P. multocida from a structural point of view. In the present work, an attempt has been made to characterize Fis by in silico methods. The structure of Fis was built by comparative modeling technique. The model of Fis was then docked onto the corresponding promoter regions of the gene encoding the capsular polysaccharide. The docked complexes of promoter DNA with Fis protein were subjected to molecular dynamics simulations to identify the mode of DNA–protein interactions. The DNA binding amino acid residues from the Fis protein were identified. And a mechanistic detail of the DNA binding interactions was predicted. So far, this is the first report that depicts the mechanistic details of Fis–DNA interactions involved in the regulation of gene expression by Fis protein. This work may therefore be useful to illuminate the still obscure molecular mechanism behind the disease propagation by P. multocida.

Carbohydrate-binding protein identification by coupling structural similarity searching with binding affinity prediction.

Carbohydrate-binding proteins (CBPs) are potential biomarkers and drug targets. However, the interactions between carbohydrates and proteins are challenging to study experimentally and computationally because of their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates as exists in RNA, DNA, and proteins. Here, we describe a structure-based function-prediction technique called SPOT-Struc that identifies carbohydrate-recognizing proteins and their binding amino acid residues by structural alignment program SPalign and binding affinity scoring according to a knowledge-based statistical potential based on the distance-scaled finite-ideal gas reference state (DFIRE). The leave-one-out cross-validation of the method on 113 carbohydrate-binding domains and 3442 noncarbohydrate binding proteins yields a Matthews correlation coefficient of 0.56 for SPalign alone and 0.63 for SPOT-Struc (SPalign + binding affinity scoring) for CBP prediction. SPOT-Struc is a technique with high positive predictive value (79% correct predictions in all positive CBP predictions) with a reasonable sensitivity (52% positive predictions in all CBPs). The sensitivity of the method was changed slightly when applied to 31 APO (unbound) structures found in the protein databank (14/31 for APO versus 15/31 for HOLO). The result of SPOT-Struc will not change significantly if highly homologous templates were used. SPOT-Struc predicted 19 out of 2076 structural genome targets as CBPs. In particular, one uncharacterized protein in Bacillus subtilis (1oq1A) was matched to galectin-9 from Mus musculus. Thus, SPOT-Struc is useful for uncovering novel carbohydrate-binding proteins. SPOT-Struc is available at http://sparks-lab.org.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.