187 Plucking the high hanging fruit: a systematic approach for targeting protein interfaces

Development of specific ligands for protein targets that help decode the complexities of protein–protein interaction networks is a key goal for the field of chemical biology. Despite the emergence of powerful in silico and experimental high-throughput screening strategies, the discovery of synthetic ligands that selectively modulate protein–protein interactions remains a challenge for the chemical biologists. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these domains, we can modulate the function of a particular protein with metabolically-stable synthetic molecules (Raj et al., 2013). This presentation will discuss computational approaches (Bullock et al., 2011; Jochim & Arora, 2010) to identify targetable interfaces along with synthetic methods (Patgiri et al., 2008; Tosovska & Arora, 2010) to develop protein domain mimics (PDMs) as modulators of intracellular protein–protein interactions (Henchey et al., 2010; Patgiri et al., 2011).     

177 T-cell vaccine design for Streptococcus pneumoniae: an in silico approach

Streptococcus pneumoniae (pneumococcus) remains an important cause of meningitis, bacteremia, acute otitis media, community acquired pneumonia associated with significant morbidity, and mortality world wide. Conjugated polysaccharide, glycoconjugated, and capsular polysaccharide based vaccines were existent for pneumococcal disease but are still specific and restricted to serotypes of S. pneumoniae. Proteome of eight serotypes of S. pneumoniae was retrieved and identified in common proteins (Munikumar et al., 2012). 18 membrane proteins were distinguished from 1657 common proteins of eight serotypes of S. pneumoniae. Implementing comparative genomic approach and subtractive genomic approach, three membrane proteins were predicted as essential for bacterial survival and non-homologous to human (Munikumar et al., 2012; Umamaheswari et al., 2011). ProPred server was used to propose four promiscuous T-cell epitopes from three membrane proteins and validated through published positive control, SYFPEITHI and immune epitope database (Munikumar et al., in press). The four epitopes docked into peptide binding region of predominant HLA-DRB alleles with good binding affinity in Maestro v9.2. The T-cell epitope 89-VVYLLPILI-97 and HLA-DRB5^?0101 docking complex was with best XPG score (?13.143?kcal/mol). Further, the stability of the complex was checked through molecular dynamics simulations in Desmond v3.3. The simulation results had revealed that the complex was stable throughout 5000?ps (Munikumar et al., in press). Thus, the epitope would be the ideal candidate for T-cell driven subunit vaccine design against selected serotypes of S. pneumoniae.     

69 DNA-binding studies of large antiviral polyamides

Polyamides are minor groove DNA-binding agents derived from the natural product distamycin A. PA1 is a large 12 ring polyamide discovered by NanoVir LLC; it is bioactive against the HPV16 virus in cell and tissue culture (Edwards et al., 2011). To better understand the basis of this phenomenon, the interactions of PA1 with the regulatory sequence of the HPV16 genome (7662–122?bp) are being examined. Using affinity cleavage as detected by capillary electrophoresis, with PA1 attached to methyl propyl ethidium iron EDTA, 10 binding sites of PA1 were identified in this part of the HPV genome. Polyamide perfect binding sites were as predicted by recognition rules (Dervan & Edelson, 2003). Quantitative DNaseI footprinting indicates that both perfect and single mismatch sites are bound with K_ds in the low nm range. Interestingly, a wide range of K_ds are observed for double mismatch sites (1–60?nm) and are under examination. This work will permit us to build a map of PA binding to HPV sequences, thus informing mechanisms of in vivo behavior.     

160 A dynamic model for the linker histone

Linker histones play an important role in the packing of chromatin. This family of proteins generally consists of a short, unstructured N-terminal domain, a central globular domain, and a C-terminal domain (CTD). The CTD, which makes up roughly half of the protein, is intrinsically disordered in solution but adopts a specific fold upon interaction with DNA (Fang et al., 2012). While the globular domain structure is well characterized, the structure of the CTD remains unknown. Sequence alignment alone does not reveal any significant homologs for this region of the protein. Construction of a model thus requires additional information. For example, the atomic model for the rat histone H1d CTD, proposed over a decade ago, used novel bioinformatics tools and biochemical data (Bharath et al., 2002). New fluorescence resonance energy transfer (FRET) studies of the folding of the CTD in the presence of linear DNA, single nucleosomes, and oligonucleosomal arrays (Caterino et al., 2011; Fang et al., 2012) have stimulated our interest in constructing a dynamic model of the protein. We have obtained preliminary information about the structure and dynamics of the linker histone CTD through ab initio folding simulations using the Rosetta modeling package (Rohl et al., 2004). By analyzing a large number of conformations sampled through a Monte Carlo procedure, we get a clearer picture of the preferred states of the protein and its dynamics. Our results show that the CTD may frequently adopt a structure with 3–5 helices and helix-turn-helix motifs in specific regions. Some of the best scoring structures show high similarity with the HMG-box-containing proteins previously used as templates by Bharath et al. Further clustering analysis of our results hints of a preferred set of conformations for the CTD of the linker histone. Comparison of these models with distances measured by FRET may help account for the distinct structures of the CTD observed upon binding to different macromolecular partners.     

161 Discovery of potent KdsA inhibitors of Leptospira interrogans through homology modeling,docking, and molecular dynamics simulations

Leptospira interrogans is the foremost cause of human leptospirosis. Discovery of novel lead molecules for common drug targets of more than 250 Leptospira serovars is of significant research interest. Lipopolysaccharide (LPS) layer prevent entry of hydrophobic agents into the cell and protect structural integrity of the bacterium. KDO-8-phosphate synthase (KdsA) catalyzes the first step of KDO biosynthesis that leads to formation of inner core of LPS. KdsA was identified as a potential drug target against Leptospira interrogans through subtractive genomic approach, metabolic pathway analysis, and comparative analysis (Amineni et al., 2010). The present study rationalizes a systematic implementation of homology modeling, docking, and molecular dynamics simulations to discover potent KdsA inhibitors (Pradhan et al., 2013; Umamaheswari et al., 2010). A reliable tertiary structure of KdsA in complex with substrate PEP was constructed based on co-crystal structure of Aquifex aeolicus KdsA synthase with PEP using Modeller9v10. Geometry-based analog search for PEP was performed from LigandInfo database to generate an in house library of 352 ligands. The ligand data-set was docked into KdsA active site through three-stage docking technique (HTVS, SP, and XP) using Glidev5.7. Thirteen lead molecules were found to have better binding affinity compared to PEP (XP Gscore?=??7.38?kcal/mol; Figure 1). The best lead molecule (KdsA- lead1 docking complex) showed XP Gscore of ?10.26?kcal/mol and the binding interactions (Figure 2) were correlated favorably with PEP–KdsA interactions (Figure 1). Molecular dynamics simulations of KdsA– lead1 docking complex for 10?ns had revealed that the complex (Figure 3) remained stable in closer to physiological environmental condition. The predicted pharmacological properties of lead1 were well within the range of a drug molecule with good ADME profile, hence, would be intriguing towards development of potent inhibitor molecule against KdsA of Leptospira.     

210 Computational study of substrates and mediators features of lacasses

Laccases are enzymes of the family multicopper oxidases, being widely used for biotechnological applications (Canas & Camarero, 2010). The enzymes’ catalytic cycle consists of the oxidation of the substrate with the concomitant reduction of molecular oxygen to water. In this process, the substrate is converted to a free radical, that can oxidize larger substrates acting as a mediator or it can undergo polymerization. Substrate binding is not specific, and there is a large diversity of substrates for laccases. Moreover, the binding site shows important differences among diverse species. The goal of the present work is to characterize the laccase binding pocket of different species, in order to establish their common pharmacophoric characteristics. For this purpose, we have carried out docking studies with a subset of substrates, covering the diversity of substrates using the Glide program (Friesner et al., 2004). We have also analyzed the characteristics of the binding site using diverse probes. We further have rationalized the differential values of km found among diverse species for a specific substrate. Finally, special attention has been devoted to the binding of the mediator 2,2′-azido-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), commonly used in industrial processes. Figure 1 shows, ABTS docked onto the fungal laccase, whereas Figure 2 shows ABTS docked onto the bacterial laccase. The analysis of the protein–ligand complex together with the corresponding optimized geometries of the possible substrate species carried out using DFT suggest that the bound species is the protonated form of ABTS as previously suggested (Enguita et al., 2004). Furthermore, the results of this study also suggest that its mechanism of oxidation occurs in a similar way to the rest of substrates/mediators, in contrast to previous suggestions (Fabbrini et al., 2002).      

172 Role of conserved water molecular triad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg 322, and Asp 364 in both the isoform of hIMPDH

Inosine monophosphate dehydrogenase (IMPDH) plays an important role in the Guanosine monophosphate (GMP) biosynthesis pathway. As hIMPDH-II is involved in CML-Cancer, it is thought to be an active target for leukemic drug design. The importance of conserved water molecules in the salt-bridge-mediated interdomain recognition and loop-flap recognition of hIMPDH has already been indicated in some simulation studies (Bairagya et al., 2009, 2011a, 2011b, 2012; Mishra et al., 2012). In this work, the role of conserved water molecules in the recognition of Inosine monophosphate (IMP) and NAD⁺ (co-factor) to active site residues of both the isoforms has been investigated by all atoms MD-Simulation studies. During 25-ns dynamics of the solvated hIMPDH-II and I (1B3O and 1JCN PDB structures), the involvement of conserved water molecular triad (W _M, W _L and W _C) in the recognition of active site residues (Asp 274, Asn 303, Arg 322, and Asp 364), IMP and NAD⁺ has been observed (Figure 1). The H-bonding co-ordination of all three conserved water molecular centers is within 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences of the water molecular interaction at the different residues (and also IMP and NAD⁺) may put forward some rational clues on antileukemic agent design.     

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).     

20 Supramolecular polymerization of nucleobase-like monomers in water

Elucidating the physiochemical principles that govern molecular self-assembly is of great importance for understanding biological systems and may provide insight into the emergence of the earliest macromolecules of life, an important challenge facing the RNA World hypothesis. Self-assembly results from a delicate balance between multiple noncovalent interactions and solvent effects, but achieving efficient self-assembly in aqueous solution with synthetic molecules has proven particularly challenging. Here, we demonstrate how two physical properties – monomer solubility and large hydrophobic surfaces of intermediate structures – are key elements to achieving supramolecular polymers in aqueous solution (Cafferty et al., 2013). Applying these two principles, we report the highly cooperative self-assembly of two weakly interacting, low molecular weight monomers [cyanuric acid and a modified triaminopyrimidine] into a water-soluble supramolecular assembly (see scheme below). The observed equilibrium between only two appreciably populated states – free monomers and supramolecular assemblies – is in excellent agreement with the values previously determined for the free energy of hydrogen bonding (Klostermeier & Millar, 2002), π???π stacking (Frier et al., 1985), and the calculated free energy penalty for the solvation of hydrophobic structures in water (Chandler, 2005). The similarity of the molecules used in this study for the nucleobases found in contemporary nucleic acids and the demonstration that these monomers assemble while the natural nucleobases do not, suggests that the first informational polymers may have emerged from a similar self-assembly process, if the nucleobases were different then they are today (Hud et al., 2013).     

The structural insights of 16.3 kDa heat shock protein (HSP16.3) from Mycobacterium tuberculosis via in silico study

Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing to tuberculosis (TB). One of the major challenges of TB management is the identification of patients, making TB diagnosis critical for disease management. This study focuses on the 16 kDa heat shock protein (HSP16.3; a potential biomarker for latent TB infection) that is expressed during the latent phase of Mtb growth. In order to explore the dynamics and interactions of HSP16.3, the 3-D structure of HSP16.3 was built via comparative modelling. The predicted structure shows a predominantly beta-sheet dodecamer with alpha-helical folds at its N-terminal. A known protein-hydrophobic probe (1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic acid; bisANS) was docked to the HSP16.3 model. Interacting residues predicted from docking and MD simulations are in good accordance with experimental data reported in the literature. MMPBSA calculation from MD simulation also showed favourable binding free energy of ?29.90 kcal/mol, driven mainly by van der waals and non-polar solvation energies. The statistical evaluation and results from the computational study on HSP16.3 indicate the reliability of the built model, which is potentially useful for further structural studies of HSP16.3 for latent TB diagnostics.     

CFTR: Domains, Structure, and Function

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.     

167 Network properties of decoy and CASP predicted models: a comparison with native protein structures

Principles that govern protein folding still remain elusive. Given the huge sequence space, it is reasonable to assume that sequences follow a particular pattern to attain one of the folds already defined in the relatively small structural space. In this study, we have used protein structure networks at different interaction strengths of non-covalent interactions (I_min) (Brinda & Vishveshwara, 2005; Kannan & Vishveshwara, 1999), to identify patterns that can distinguish a native protein from decoy/modelled structures. This is a rigorous extension of an earlier study performed at I_min???0% (Chatterjee, Bhattacharyya et al., 2012). Network properties such as the size of the largest cluster (SLClu), largest k-2 communities (ComSk2) and clustering coefficients (CCoe) are analysed for 5422 native structures and 29543 decoy/modelled structures. Steeper transition profile of the native structures as a function of I_min is consistently observed (see Figure) . The network properties generated at different I_min and main-chain hydrogen bonds (MHB) are integrated into support vector machine to build a classifier, giving an accuracy of 94.11%. The uniqueness of the protein structures through side-chain interactions are captured by the network parameters, while MHB represents the backbone packing. Quality predictions for the recently concluded CASP 10 predicted models are also performed using the model with the selected ones showing RMSD values?<?2.5?Å with respect to the native structures. Amongst the network properties, ComSk2 is maximally able to capture the transition properties of the structures. Importance of ComSk2 has earlier been implicated to capture the percolating behaviour of a protein structure (Deb & Vishveshwara, 2009). Overall, a robust classifier is obtained, and patterns specific to native structures have been analysed and discussed. The study highlights the importance of side-chain interactions at different I_mins, along with backbone level interactions.     

CRISPR/Cas系统与结核病防控新措施相关性研究

结核分枝杆菌(Mycobacterium tuberculosis,MTB,以下简称结核杆菌)感染引起的结核病仍然是严重影响人类健康全球性重大传染病。全球约1/4人口是结核杆菌的潜伏感染者。2019年,世界卫生组织(Worldhealthorganization,WHO)报道全球约150万人死于结核病。深入研究结核杆菌生物学有望为结核病防控提供新工具。成簇规律性间隔短回文重复(Clusteredregularlyinterspacedshort palindromic repeats,CRISPR)/Cas是细菌免疫系统的重要成分,在结核杆菌等分枝杆菌中也广泛存在,同时,也是分枝杆菌基因编辑的重要工具。本文结合课题组研究工作,综述了结核杆菌III-A型CRISPR/Cas系统各组分的生物学功能以及与致病的相关性,CRISPR/Cas编辑工具在诊断治疗耐药结核杆菌和结核病防控新措施中的进展。     

39 Simulation of RNA tandem GA base pairs provides insights about the forces behind conformational preference

Conformational changes are important for RNA function. We used molecular mechanics with all-atom models to understand conformational preference in RNA tandem guanine–adenine (GA) base pairs. These tandem GA base pairs play important roles in determining the stability and structural dynamics of RNA tertiary structures. Previous solution structures showed that these tandem GA base pairs adopt either imino (cis-Watson-Crick/cis-Watson-Crick interaction) or sheared (trans-Hoogsteen/trans-Hoogsteen interaction) pairing depending upon the sequence and orientation of the adjacent base pairs. In our simulations, we modeled (GCGGACGC)₂ (Wu and Turner 1996) and (GCGGAUGC)₂ (Tolbert et al., 2007), experimentally preferred as imino and sheared, respectively. Besides the experimentally preferred conformation, we constructed models of the nonnative conformations by changing cytosine to uracil or uracil to cytosine. We used explicit solvent molecular dynamics and free energy calculation with umbrella sampling to measure the free energy deference of the experimentally preferred conformation and the nonnative conformations. A modification to ff10 was required, which allowed the guanine bases’ amino group to leave the base plane (Yildirim et al., 2009). With this modification, the RMSD of unrestrained simulations and the free energy surfaces are improved, suggesting the importance of electrostatic interactions by G amino groups in stabilizing the native structures.     

Eukaryotic-like adenylyl cyclases in Mycobacterium tuberculosis H37Rv: cloning and characterization.

Screening the Mycobacterium tuberculosis H37Rv genomic library for complementation of catabolic defect for cAMP-dependent expression of maltose operon produced the adenylyl cyclase gene (Mtb cya, (1997)) annotated later as Rv1625c (Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, et al. (1998) Nature 393, 537-544). The deduced amino acid (aa) sequence (443 aa) encoded by Mtb cya contains a single hydrophobic domain of six transmembrane helices (152 aa) in the amino-terminal half of the protein. Flanking this domain are an arginine-rich (17%) amino-terminal cytoplasmic tail (46 aa) and a carboxyl-terminal cytoplasmic domain (245 aa) with extensive homology to the catalytic core of eukaryotic adenylyl cyclases. Site-directed mutagenesis of Arg(43) and Arg(44) to alanine/glycine showed a loss of adenylyl cyclase activity, whereas mutagenesis to lysine restored the activity. Hence it is proposed that the formation of the catalytic site in Mtb adenylyl cyclase requires an interaction between Arg(43) and Arg(44) residues in the distal cytoplasmic tail and the carboxyl-terminal cytoplasmic domain. Mtb adenylyl cyclase activity at the physiological concentration of ATP (1 mm) was 475 nmol of cAMP/min/mg of membrane protein in the presence of Mn(2+) but only 10 nmol of cAMP/min/mg of membrane protein in the presence of Mg(2+). The physiological significance of the activation of Mtb adenylyl cyclase by Mn(2+) is discussed in view of the presence of manganese transporter protein in mycobacteria and macrophages wherein mycobacteria reside.