Site Binding Mode Research Articles

Purification, bio-chemical characterization, homology modeling and active site binding mode interactions of thermo-alkali-tolerant β-1,4 endoxylanase from Coprinus cinereus LK-D-NCIM-1369

Abstract Present study aims at purifying and characterizing crude xylanase isolated from Coprinus cinereus LK-D-NCIM-1369 under solid-state fermentation conditions. Crude xylanase is purified by (NH4) 2 SO 4 precipitation, carboxymethyl cellulose cation-exchange and gel filtration chromatography with Superdex-200 column. Purified xylanase from C. cinereus shows 54.3% similarity with β-1,4 endoxylanase from C. cinerea okayama7#130 with accession number gi|169855830 based on MALDI-TOF/TOF. The purified xylanase—a monomeric protein with molecular weight 20.1 kD shows maximum activity at 60 °C and pH 7.0 and stable over pH 5.0–9.0 and temperature up to 70 °C. The xylanase shows K m and V values of 3.26 mg/mL and 909.09 µm/min/mg for birchwood xylan. A sequence of 186 amino acids of C. cinerea okayama7#130 gi|169855830 is retrieved from NCBI database and its 3-D model is generated on the basis of crystal structure of xylanase 1XNK-A from Chaetomium thermophilum with the help of online server SWISS-MODEL workspace. The model is verified and validated on SAVES and PROCHECK programmes, respectively. Ramachandran plot reveals that the total residues in allowed and generously allowed regions are 99.4% and 0.6% respectively. Overlapping of xylanase with the template of 1XNK-A stipulates the amino acid residues Asn35, Tyr68, Arg113, Ser118, Tyr168 and Glu174 constitute active site of the enzyme.

Molecular Modeling and Active Site Binding Mode Characterization of Aspartate β-Semialdehyde Dehydrogenase Family.

The enzyme aspartate β-semialdehyde dehydrogenase (ASADH) plays a vital role in biosynthesis of essential amino acids and several important metabolites in microbes and some higher plants. So this key enzyme can be targeted selectively in these microbes to exhibit anti-bacterial and fungicidal effects. In this work, molecular modeling and comparative active site binding mode studies were performed for understanding the mode of action, in silico insight into the 3D structure, enzyme-substrate interactions with natural substrate in this homologous enzyme family. During comparative sequence analysis, high diversity was found in the sequences of different ASADHs and exhibited the same key binding interactions with the substrate. Both, the functional carboxylic and the phosphate group of the substrate are engaged in a bidentate interaction with the guanidinium N atom of two key arginyl active site residues of ASADHs. These structural and active site binding mode characterization studies can further be used for designing the more potent and selective substrate analogues inhibitors against ASADH family.

Mechanistic insights into the hydrolysis of organophosphorus compounds by paraoxonase-1: exploring the limits of substrate tolerance in a promiscuous enzyme

We designed, synthesized and screened a library of analogs of the organophosphate pesticide metabolite paraoxon against a recombinant variant of human serum paraoxonase-1. Alterations of both the aryloxy leaving group and the retained alkyl chains of paraoxon analogs resulted in substantial changes to binding and hydrolysis, as measured directly by spectrophotometric methods or in competition experiments with paraoxon. Increases or decreases in the steric bulk of the retained groups generally reduced the rate of hydrolysis, while modifications of the leaving group modulated both binding and turnover. Studies on the hydrolysis of phosphoryl azide analogs as well as amino-modified paraoxon analogs, the former being developed as photo-affinity labels, found enhanced tolerance of structural modifications, when compared with O-alkyl substituted molecules. Results from computational modeling predict a predominant active site binding mode for these molecules which is consistent with several proposed catalytic mechanisms in the literature, and from which a molecular-level explanation of the experimental trends is attempted. Overall, the results of this study suggest that while paraoxonase-1 is a promiscuous enzyme, there are substantial constraints in the active site pocket, which may relate to both the leaving group and the retained portion of paraoxon analogs.

Protein modeling and active site binding mode interactions of myrosinase–sinigrin in Brassica juncea—An in silico approach

Myrosinase, the only known S-glycosidase, occurs particularly in Cruciferae family. It is responsible for the hydrolysis of glucosinolates and serves as a vital element of plant defense system. The biological and chemical properties of myrosinase catalyzed products of glucosinolates are well characterized. The myrosinase-protein-sequence of Brassica juncea was retrieved from NCBI database and its 3-D model was generated on the basis of crystal structure of 1MYR-A, 1E4M-M and 1DWA-M chains of myrosinase from Sinapis alba by employing Modeller9v7 program. Homolog templates from S. alba exhibited 72% identity with target sequence. The model was optimized by using molecular dynamics (MD) approach together with simulated annealing (SA) methods in the same Modeller program, and eventually verified and validated on SAVES (Structure Analysis and Verification Server) and PROCHECK programs, respectively. Ramachandran plot obtained through PROCHECK program depicted that 99.8% of total residues were confined to the allowed region while only one residue (Thr92) was restrained to the disallowed region. Additionally, B. juncea myrosinase contains three disulphide bridges which were found to be conserved in S. alba homologs as well. Further, overlapping of B. juncea myrosinase with that of template protein 1MYR-A from S. alba stipulates the amino acid residues Arg115, Gln207, Thr210, Asn350, Tyr352 and Glu429 that constitute active site of the enzyme. Active site analysis also speculates the presence of a hydrophobic pocket in addition to seven N-glycosylation sites. Docking studies of enzyme and substrate illuminate the interactions of various active site residues with diverse groups of sinigrin. Therefore, the present study furnishes the first significant, in silico insight into the 3-D structure, active site machinery, and enzyme–substrate interactions of B. juncea myrosinase.

Discovery of 6-(Aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamides as Potent, Selective Dipeptidyl Peptidase-4 (DPP4) Inhibitors

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.

Active site binding modes of dimeric phloroglucinols for HIV-1 reverse transcriptase, protease and integrase

In a recent report on anti-HIV activity of dimeric phloroglucinol compounds, seven out of twenty one compounds were shown to possess good activity in HIV infected human CD4+ T cell line. The seven active compounds were docked into the active sites of HIV-1 reverse transcriptase (RT), integrase (IN), and protease (PR). Two compounds which have RT inhibitory activity exhibited H-bonding interaction with Lys101. Compounds 1, 5, and 6 exhibited good binding interactions with catalytic residues Asp64 and Asp116, while compounds 5 and 7 showed binding with PR (Asp25, Gly27, and Asp29). We propose here that compound 5 may be a dual inhibitor acting against both IN and PR. The docking results gave the information about active site binding modes of dimeric phloroglucinols interacting with HIV proteins.

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride

Background We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. Methods The combination of experimental and theoretical methods was used. Results The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by 31P NMR experiments. The thermodynamics of the ligand–nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. Conclusions Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. General significance The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein–DNA interactions. However, these are not clear for the DNA–small molecule interactions and we believe that our studies will bring a new insight into such phenomena.

Interactions of curcumin with the PfATP6 model and the implications for its antimalarial mechanism

Despite curcumin has been proved to possess antimalarial effects, the underlying mechanism remains to be elucidated. In this letter, the active site binding modes of curcumin in PfATP6, an important antimalarial target, were investigated using computational docking. It was revealed that curcumin interacts with PfATP6 mainly through hydrophobic interactions and hydrogen bonds. Moreover, the theoretically predicted binding affinity implies that curcumin can efficiently inhibit PfATP6, which gains some deeper insights into the antimalarial mechanism of curcumin.

Protonation state and substrate binding to B2 metallo‐β‐lactamase CphA from Aeromonas hydrofila

The zinc enzymes metallo beta-lactamases counteract the beneficial action of beta-lactam antibiotics against bacterial infections, by hydrolyzing their beta-lactam rings. To understand structure/function relationships on a representative member of this class, the B2 M beta L CphA, we have investigated the H-bond pattern at the Zn enzymatic active site and substrate binding mode by molecular simulation methods. Extensive QM calculations at the DFT-BLYP level on eleven models of the protein active site, along with MD simulations of the protein in aqueous solution, allow us to propose two plausible protonation states for the unbound enzyme, which are probably in equilibrium. Docking procedures along with MD simulations and QM calculations suggest that in the complex between the enzyme and its substrate (biapenem), the latter is stable in only one of the two protonation states, in addition it exhibits two different binding modes, of which only one agrees with previous proposals. In both cases, the substrate is polarized as in aqueous solution. We conclude that addressing mechanistic issues on this class of enzymes requires a careful procedure to assign protonation states and substrate docking modes.

Correlation of Biological Activity with Active Site Binding Modes of Geminal Disulfone HIV‐1 Integrase Inhibitors

Binding modes of a series of potent geminal disulfone-containing HIV-1 integrase inhibitors were investigated using AutoDock 3.0. Two major conformations were shared for this series of compounds: a “U-shaped” conformation and an “L-shaped” conformation. The data presented suggest that the U-shaped conformer may be the more biologically relevant conformation and that the design of molecules that are structurally biased in that conformation could lead to more potent analogues.

Biophysical Characterization of the Sterol Demethylase P450 from Mycobacterium tuberculosis, Its Cognate Ferredoxin, and Their Interactions

Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An epsilon419 of 134 mM(-1) cm(-1) was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)-CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min(-1) at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450-P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (Kd = 21.7 microM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at approximately 558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron-sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is -375 mV, increasing to -225 mV in the estriol-bound form. The Fdx potential is -31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min(-1) in the ligand-free form and 4.3 min(-1) in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.

Active site binding modes of curcumin in HIV-1 protease and integrase

Structure models for the interaction of curcumin with HIV-1 integrase (IN) and protease (PR) were investigated using computational docking. Curcumin was found to bind preferentially in similar ways to the active sites of both IN and PR. For IN, the binding site is formed by residues Asp64, His67, Thr66, Glu92, Thr93, Asp116, Ser119, Asn120, and Lys159. Docked curcumin contacts the catalytic residues adjacent to Asp116 and Asp64, and near the divalent metal (Mg 2+). In the PR docking, the curcumin structure fitted well to the active site, interacting with residues Asp25, Asp29, Asp30, Gly27′, Asp29′, and Asp30′. The results suggest that o-hydroxyl and/or keto–enol structures are important for both IN and PR inhibitory actions. The symmetrical structure of curcumin seems to play an important role for binding to the PR protein, whereas the keto–enol and only one side of the terminal o-hydroxyl showed tight binding to the IN active site.

Active site binding modes of the β-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design

Predicting a bioactive conformation of a ligand is of paramount importance in rational drug design. The task becomes very difficult when the receptor site possesses a region with unusual conformational flexibility. Significant conformational differences are present in the active site regions in the available crystal structures of the core domains of HIV-1 integrase (IN). Among all reported IN inhibitors, the β-diketoacid class of compounds has proved to be of most promise and indeed S-1360 was the first IN inhibitor to enter clinical studies. With an aim to predict the bioactive (active site bound) conformation of S-1360, we performed extensive docking studies using three different reported crystal structures where the active site or partial active site region was resolved. For comparison we extended our studies to include 5CITEP (the first compound cocrystallized with IN core domain) and a bis-diketoacid (BDKA). We found that the conformation of S-1360 when bound in one of the active sites matches that of the experimentally observed results of IN escape mutants resistant to S-1360. Therefore, we propose that this active site conformation is the biologically relevant conformation and can be used for the future structure-based drug design studies selectively targeting IN.

Chemistry and Biology of Deoxy-myo-inositol Phosphates: Stereospecificity of Substrate Interactions within an Archaeal and a Bacterial IMPase

Six enantiomerically pure myo-inositol-monophosphates, including four deoxygenated analogues, have been synthesized by employing catalytic asymmetric phosphorylation methodology. These compounds were then evaluated as substrates for the direct interrogation of the stereospecificity of enzyme-substrate interactions with two inositol-monophosphatases (IMPases), one of which (from Archaeoglobus fulgidus) is characterized by an X-ray crystal structure with its substrate (d-I-1P) bound. The kinetic results lead to the finding that certain hydroxyl group contacts are actually destabilizing, while others have little effect. These new probes also allow a prediction of the active site binding mode of the substrate for the Escherichia coli IMPase for which no crystal structure exists.

Binding modes of 6,7 di-substituted 4-anilinoquinoline-3-carbonitriles to EGFR

4-Anilino-3-cyanoquinolines were reported to have irreversible binding to epidermal growth factor receptor kinase (EGFRK) by forming a covalent linkage to C773. Our initial docking studies gave results inconsistent with the in vitro data and showed two different binding modes. To perceive the exact mode of binding of these ligands, two models of the ligand–EGFR complexes were considered: (1) reversible binding mode in which the ligand had hydrogen bond interactions at the binding site and (2) irreversible binding mode wherein the ligand's Michael acceptor side chain has proximity to the sulfhydryl group of C773 of EGFR, thereby enabling a covalent interaction. The irreversible binding mode correlated better than reversible binding mode with respect to in vitro data. However, our results indicate that both modes are being adopted by the ligands and could be utilized to design more potent EGFRK inhibitors.

Interactions of the antibiotics neomycin B and chlortetracycline with the hammerhead ribozyme as studied by Zn2+-dependent RNA cleavage

We have investigated the interactions of two antibiotics, neomycin B and chlortetracycline (CTC), with the hammerhead ribozyme using two Zn2+ cleavage sites at U4 and A9 in its catalytic core. CTC-dependent inhibition of Zn2+ cleavage was observed in all cases. In contrast, we unexpectedly observed acceleration of A9 cleavage by neomycin under low ionic strength conditions similar to those used to study inhibition of hammerhead substrate cleavage by this antibiotic. This result provides evidence that the inhibitory mechanism of neomycin does not include competition with the metal ion bound to the A9/G10.1 metal-ion binding site, as previously proposed. Under high ionic strength conditions, optimized for Zn2+-dependent cleavage, we observed neomycin-dependent inhibition of cleavage at both A9 and U4. The ability of neomycin to both inhibit and accelerate Zn2+ cleavage suggests that there is either more than one neomycin binding site or multiple binding modes at a single site in the hammerhead ribozyme. Furthermore, the accessibilities and/or affinities of disparate neomycin binding sites or binding modes are dependent on the ionic strength and the pH of the medium.

Characterization of the electrophile binding site and substrate binding mode of the 26‐kDa glutathione S‐transferase from Schistosoma japonicum

The 26-kDa glutathione S-transferase from Schistosoma japonicum (Sj26GST), a helminth worm that causes schistosomiasis, catalyzes the conjugation of glutathione with toxic secondary products of membrane lipid peroxidation. Crystal structures of Sj26GST in complex with glutathione sulfonate (Sj26GSTSLF), S-hexyl glutathione (Sj26GSTHEX), and S-2-iodobenzyl glutathione (Sj26GSTIBZ) allow characterization of the electrophile binding site (H site) of Sj26GST. The S-hexyl and S-2-iodobenzyl moieties of these product analogs bind in a pocket defined by side-chains from the beta1-alpha1 loop (Tyr7, Trp8, Ile10, Gly12, Leu13), helix alpha4 (Arg103, Tyr104, Ser107, Tyr111), and the C-terminal coil (Gln204, Gly205, Trp206, Gln207). Changes in the Ser107 and Gln204 dihedral angles make the H site more hydrophobic in the Sj26GSTHEX complex relative to the ligand-free structure. These structures, together with docking studies, indicate a possible binding mode of Sj26GST to its physiologic substrates 4-hydroxynon-2-enal (4HNE), trans-non-2-enal (NE), and ethacrynic acid (EA). In this binding mode, hydrogen bonds of Tyr111 and Gln207 to the carbonyl oxygen atoms of 4HNE, NE, and EA could orient the substrates and enhance their electrophilicity to promote conjugation with glutathione.

Active site binding modes of HIV-1 integrase inhibitors.

Using the crystal structure of the first complex of the HIV-1 integrase catalytic core domain with an inhibitor bound to the active site, structural models for the interaction of various inhibitors with integrase were generated by computational docking. For the compound of the crystallographic study, binding modes unaffected by crystal packing have recently been proposed. Although a large search region was used for the docking simulations, the ligands investigated here are found to bind preferably in similar ways close to the active site. The binding site is formed by residues 64-67, 116, 148, 151-152, 155-156, and 159, as well as by residue 92 in case of the largest ligand of the series. The coherent picture of possible interactions of small-molecule inhibitors at the active site provides an improved basis for structure-based ligand design. The recurring motif of tight interaction with the two lysine residues 156 and 159 is suggested to be of prime importance.

Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography.

The three-dimensional structures of bovine lens leucine aminopeptidase (blLAP) complexed with L-leucinal and of the unliganded enzyme have been determined at crystallographic resolutions of 1.9 and 1.6 A, respectively. Leucinal binds as a hydrated gem-diol to the active site of b1LAP), resembling the presumed gem-diolated intermediate in the catalytic pathway. One hydroxyl group bridges the two active site metal ions, and the other OH group is coordinated to Zn1. The high-resolution structure of the unliganded enzyme reveals one metal-bound water ligand, which is bridging both zinc ions. Together, these structures support a mechanism in which the bridging water ligand is the attacking hydroxide ion nucleophile. The gem-diolate intermediate is probably stabilized by four coordinating bonds to the dizinc center and by interaction with Lys-262 and Arg-336. In the mechanism, Lys-262 polarizes the peptide carbonyl group, which is also coordinated to Zn1. The Arg-336 side chain interacts with the substrate and the gem-diolate intermediate via water molecules. Near Arg-336 in the b1LAP-leucinal structure, an unusually short hydrogen bond is found between two active site water molecules.