Structures and binding studies of the complexes of phospholipase A2 with five inhibitors.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipids into arachidonic acid and lysophospholipids. Arachidonic acid is used as a substrate in the next step of the multistep pathway leading to the production of eicosanoids. The eicosanoids, in extremely low concentrations, are required in a number of physiological processes. However, the increase in their concentrations above the essential physiological requirements leads to various inflammatory conditions. In order to prevent the unwanted rise in the concentrations of eicosanoids, the actions of PLA2 and other enzymes of the pathway need to be blocked. We report here the structures of five complexes of group IIA PLA2 from Daboia russelli pulchella with tightly binding inhibitors, (i) p-coumaric acid, (ii) resveratrol, (iii) spermidine, (iv) corticosterone and (v) gramine derivative. The binding studies using fluorescence spectroscopy and surface plasmon resonance techniques for the interactions of PLA2 with the above five compounds showed high binding affinities with values of dissociation constants (KD) ranging from 3.7×10(-8) M to 2.1×10(-9) M. The structure determinations of the complexes of PLA2 with the above five compounds showed that all the compounds bound to PLA2 in the substrate binding cleft. The protein residues that contributed to the interactions with these compounds included Leu2, Leu3, Phe5, Gly6, Ile9, Ala18, Ile19, Trp22, Ser23, Cys29, Gly30, Cys45, His48, Asp49 and Phe106. The positions of side chains of several residues including Leu2, Leu3, Ile19, Trp31, Lys69, Ser70 and Arg72 got significantly shifted while the positions of active site residues, His48, Asp49, Tyr52 and Asp99 were unperturbed.

Heterogeneity and antibiotic resistance in Propionibacterium acnes isolates and its therapeutic implications: blurring the lines between commensal and pathogenic phylotypes.

Acne vulgaris is a multifactorial skin disease associated with the colonization of Propionibacterium acnes. Antibiotics are a mainstay of treatment for acne, yet the emergence of resistance against the currently approved antibiotics is a serious concern. In this case report, a slow responder had multiple Propionibacterium acnes isolates with varied levels of sensitivity to the conventional antibiotics. The bacterial isolates obtained from acne samples collected from the patient were analyzed for phylogeny, and was found to be largely restricted to two different lineage patterns. Propionibacterium acnes phylotype IA1, which is considered to be pathogenic, displayed clindamycin sensitivity, but phylotype IB, which is associated with commensals, exhibited high clindamycin resistance. Sensitivity analysis revealed uniform resistance to macrolides, but susceptibility to tetracycline and nadifloxacin. These results implicate Propionibacterium acnes in the pathophysiology of acne vulgaris, although the lines between commensal and pathological phylotypes may be blurred. Switching the patient to a combination of minocycline and nadifloxacin resulted in a significant improvement in the clinical lesions. Such a science-driven judicious selection of antibiotics can minimize the probability of development of resistance, and might be the way forward in the treatment of acne.

Antibiotic-resistant acne: getting under the skin.

Propionibacterium acnes is a key pathogenic factor in the development of acne. Antibiotics are the first choice of treatment for mild-to-moderate, mixed, papular/pustular, and moderate nodular acne, and an alternative choice in severe, nodular/conglobate acne. The emergence of resistance to the currently available antibiotics poses a serious set-back to this algorithm, and the reduced arsenal can diminish efficacy of treatment. This emerging situation should catalyze innovations in dermatology; for example, newer drugs and technologies such as next-generation antibiotics with excellent potency and low propensity to develop resistance, rapid diagnostic platforms to select responders and nonresponders, and delivery technologies that target the bacteria. Such innovations can dramatically expand the arsenal for dermatologists in the management of acne.

Structural and functional insights into peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.

Structural and binding studies of peptidyl-tRNA hydrolase from Pseudomonas aeruginosa provide a platform for the structure-based inhibitor design against peptidyl-tRNA hydrolase.

During the course of protein synthesis in the cell, the translation process is often terminated due to various reasons. As a result, peptidyl-tRNA molecules are released which are toxic to the cell as well reducing the availability of free amino acid and tRNA molecules for the required protein synthesis in the cell. Such a situation is corrected by an enzyme, Pth (peptidyl-tRNA hydrolase), which catalyses the release of free tRNA and peptide moieties from peptidyl-tRNAs. This means that the active Pth is essential for the survival of bacteria. In order to design inhibitors of PaPth (Pth from Pseudomonas aeruginosa), we determined the structures of PaPth in its native and bound states with compounds amino acylate-tRNA analogue and 5-azacytidine. The structure determination of the native protein revealed that the substrate-binding site was partially occupied by Glu161 from the neigh-bouring molecule. The structure of PaPth indicated that the substrate-binding site can be broadly divided into three distinct subsites. The structures of the two complexes showed that the amino acylate-tRNA analogue filled three subsites, whereas 5-azacytidine filled two subsites. The common sugar and the base moieties of the two compounds occupied identical positions in the cleft. Using surface plasmon resonance, the dissociation constants for the amino acylate-tRNA analogue and 5-azacytidine were found to be 3.53×10-8 M and 5.82×10-8 M respectively.

Current overview of allergens of plant pathogenesis related protein families.

Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states.

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67Å, 1.60Å, 2.20Å, 1.70Å, 2.07Å and 1.90Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.

First structural evidence of sequestration of mRNA cap structures by type 1 ribosome inactivating protein from Momordica balsamina.

This is the first structural evidence of recognition of mRNA cap structures by a ribosome inactivating protein. It is well known that a unique cap structure is formed at the 5' end of mRNA for carrying out various processes including mRNA maturation, translation initiation, and RNA turnover. The binding studies and crystal structure determinations of type 1 ribosome inactivating protein (RIP-1) from Momordica balsamina (MbRIP-1) were carried out with mRNA cap structures including (i) N7-methyl guanine (m7G), (ii) N7-methyl guanosine diphosphate (m7GDP), and (iii) N7-methyl guanosine triphosphate (m7GTP). These compounds showed affinities to MbRIP-1 at nanomolar concentrations. The structure determinations of the complexes of MbRIP-1 with m7G, m7GDP, and m7GTP at 2.65, 1.77, and 1.75 Å resolutions revealed that all the three compounds bound to MbRIP-1 in the substrate binding site at the positions which are slightly shifted towards Glu85 as compared to those of rRNA substrates. In this position, Glu85 forms several hydrogen bonds with guanine moiety while N-7 methyl group forms van der Waals contacts. However, the guanine rings are poorly stacked in these complexes. Thus, the mode of binding by MbRIP-1 to mRNA cap structures is different which results in the inhibition of depurination. Since some viruses are known to exploit the capping property of the host, this action of MbRIP-1 may have implications for the antiviral activity of this protein in vivo. The understanding of the mode of binding of MbRIP-1 to cap structures may also assist in the design of anti-viral agents.

Structural basis of the binding of fatty acids to peptidoglycan recognition protein, PGRP-S through second binding site.

Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5) to 10(-8) M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN-γ by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.

Cloning, expression, crystallization and preliminary structural studies of dihydrodipicolinate reductase from Acinetobacter baumannii.

Acinetobacter baumannii is a virulent pathogenic bacterium that is resistant to most currently available antibiotics. Therefore, the design of drugs for the treatment of infections caused by A. baumannii is urgently required. Dihydrodipicolinate reductase (DHDPR) is an important enzyme which is involved in the biosynthetic pathway that leads to the production of L-lysine in bacteria. In order to design potent inhibitors against this enzyme, its detailed three-dimensional structure is required. DHDPR from A. baumannii (AbDHDPR) has been cloned, expressed, purified and crystallized. Here, the preliminary X-ray crystallographic data of AbDHDPR are reported. The crystals were grown using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitating agent The crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 80.0, b = 100.8, c = 147.6 Å, and contained four molecules in the asymmetric unit. The complete structure determination of AbDHDPR is in progress.

Current perspectives in NSAID-induced gastropathy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most highly prescribed drugs in the world. Their analgesic, anti-inflammatory, and antipyretic actions may be beneficial; however, they are associated with severe side effects including gastrointestinal injury and peptic ulceration. Though several approaches for limiting these side effects have been adopted, like the use of COX-2 specific drugs, comedication of acid suppressants like proton pump inhibitors and prostaglandin analogs, these alternatives have limitations in terms of efficacy and side effects. In this paper, the mechanism of action of NSAIDs and their critical gastrointestinal complications have been reviewed. This paper also provides the information on different preventive measures prescribed to minimize such adverse effects and analyses the new suggested strategies for development of novel drugs to maintain the anti-inflammatory functions of NSAIDs along with effective gastrointestinal protection.

Treatment recalcitrant cases of tinea corporis/cruris caused by T. mentagrophytes - interdigitale complex with mutations in ERG11 ERG 3, ERG4, MDR1 MFS genes & SQLE and their potential implications.

BACKGROUND: Recalcitrant dermatophyte infections are being reported from various parts of the world due to varied causes including strain variation, steroid misuse, SQLE mutations, and variable quality of itraconazole pellet formulations. The oral drug preferred in endemic areas is itraconazole, to which MIC levels remain low, and clinical failures to itraconazole reported defy a sound scientific explanation. OBJECTIVES: The objective of the study was to conduct a proteomic and genomic analysis on isolates from therapeutically recalcitrant case with isolation of gene mutations and enzymatic abnormalities to explain azole failures. METHODS: Trichophyton mentagrophyte interdigitale complex strains were isolated from seven clinically non-responding tinea corporis/cruris patients, who had failed a sequential course of 6 weeks of terbinafine 250 mg QD and itraconazole 100 mg BID. After AFST 1 strain, KA01 with high MIC to most drugs was characterized using whole genome sequencing, comparative proteomic profiling, and total sterol quantification. RESULTS: Sterol quantification showed that the standard strain of Trichophyton mentagrophytes (MTCC-7687) had half the ergosterol content than the resistant KA01 strain. Genomic analysis revealed mutations in SQLE, ERG4, ERG11, MDR1, MFS genes, and a novel ERG3 mutation. Proteomic analysis established the aberrant expression of acetyl Co-A transferase in the resistant strain and upregulation of thioredoxin reductase and peroxiredoxin. CONCLUSION: Our findings demonstrate possible reasons for multidrug resistance in the prevalent strain with mutations in genes that predict terbinafine (SQLE) and azole actions (ERG4, ERG11, ERG3) apart from efflux pumps (MDR1, MFS) that can explain multidrug clinical failures.

Multiligand specificity of pathogen-associated molecular pattern-binding site in peptidoglycan recognition protein.

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and ß-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10(-7) M, whereas the binding affinities for GlcNAc and ß-maltose separately are in the range of 10(-4) M to 10(-5) M, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10(-6) M. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and ß-maltose revealed that MDP, GlcNAc, and ß-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and ß-maltose occupy distinct non-overlapping positions belonging to different subsites.

Structural basis of recognition of pathogen-associated molecular patterns and inhibition of proinflammatory cytokines by camel peptidoglycan recognition protein.

Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10(-9) and 2.4 × 10(-8) M, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.

A potent antibiotic-loaded bone-cement implant against staphylococcal bone infections.

New antibiotics should ideally exhibit activity against drug-resistant bacteria, delay the development of bacterial resistance to them and be suitable for local delivery at desired sites of infection. Here, we report the rational design, via molecular-docking simulations, of a library of 17 candidate antibiotics against bone infection by wild-type and mutated bacterial targets. We screened this library for activity against multidrug-resistant clinical isolates and identified an antibiotic that exhibits potent activity against resistant strains and the formation of biofilms, decreases the chances of bacterial resistance and is compatible with local delivery via a bone-cement matrix. The antibiotic-loaded bone cement exhibited greater efficacy than currently used antibiotic-loaded bone cements against staphylococcal bone infections in rats. Potent and locally delivered antibiotic-eluting polymers may help address antimicrobial resistance.

Mode of binding of the tuberculosis prodrug isoniazid to heme peroxidases: binding studies and crystal structure of bovine lactoperoxidase with isoniazid at 2.7 A resolution.

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe(422) O, Gln(423) O(epsilon1), and Phe(254) O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe(3+), His(109) N(epsilon2), and Gln(105) N(epsilon2). The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C(beta) and C(gamma) atoms of Glu(258), and C(gamma) and C(delta) atoms of Arg(255). The binding studies indicate that INH binds to LPO with a value of 0.9 x 10(-6) m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H(2)O(2) with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.

Structure of the novel 14kDa fragment of alpha-subunit of phycoerythrin from the starving cyanobacterium Phormidium tenue.

The rod-like phycobilisome (PBS) in cyanobacterium is the light-harvesting complex of phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). The orderly degradation of PBS was observed under starvation conditions. A 14 kDa truncated fragment of alpha-subunit of PE (F-alphaPE) was identified from the degraded product. F-alphaPE was purified to homogeneity, sequenced and crystallized. The merohedrally twinned crystals with a twinning factor of approximately 0.5 were obtained. The crystal structure of F-alphaPE was determined with molecular replacement method using detwinned data and refined to an R(cryst) factor of 23.2% (R(free)=27.6%). The structure consisted of two crystallographically independent molecules in the asymmetric unit. The two molecules were designated as molecules A and B with a buried area of 200 A(2) at the interface. The structure of F-alphaPE consists of seven alpha-helices A, B, E, F, F', G and H. The first 31N-terminal residues that fold into parallel alpha-helices X and Y in other PEs are not present in the amino acid sequence of F-alphaPE. Both molecules, A and B contain two chromophore ligands, PEB1 and PEB2 in each. These are covalently linked to the polypeptide chain through Cys82 and Cys139, respectively. The superimposition of C(alpha) tracings of molecules A and B shows an r.m.s. shift of 1.0 A indicating that the structures of two independent molecules are very similar. The degradation of phycobilisome proteins under starvation stress seems to occur to supplement the requirement of amino acids for protein synthesis and to reduce the absorption of light energy.

Modulation of inhibitory activity of xylanase-α-amylase inhibitor protein (XAIP): binding studies and crystal structure determination of XAIP-II from Scadoxus multiflorus at 1.2 Å resolution.

BACKGROUND: Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C) was reported to inhibit two structurally unrelated enzymes xylanase GH11 and α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II) that inhibits α-amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature. RESULTS: In order to determine the structural basis of the enhanced potency of XAIP-II against α-amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR). CONCLUSION: The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop α3-ß3 (residues, 102 - 118) which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206), α7 (residues, 230 - 243) and loop ß6-α6 (residues, 180 - 192) adopts a stereochemically more favorable conformation due to replacements of residues, Ser190, Gly191 and Glu194 by Ala191, Ser192 and Ser195 respectively in α-helix α6, Glu231 and His236 by Thr232 and Ser237 respectively in α-helix α7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with α-amylase GH13 as compared to XAIP. These observations correlate well with the values of 4.2 × 10(-6) M and 3.4 × 10(-8) M for the dissociation constants of XAIP-II with xylanase GH11 and α-amylase GH13 respectively and those of 4.5 × 10(-7) M and 3.6 × 10(-6) M of XAIP with xylanase GH11 and α-amylase GH13 respectively.

First structural evidence for the mode of diffusion of aromatic ligands and ligand-induced closure of the hydrophobic channel in heme peroxidases.

The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole (amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules, including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H2O2 to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic channel as well as the binding of H2O2. This explains the amitrole toxicity. From binding studies, the dissociation constant (Kd) for amitrole with lactoperoxidase was found to be approximately 5.5x10(-7) M, indicating high affinity.