Structural insight into the inhibition of acetylcholinesterase by 2,3,4, 5-tetrahydro-1, 5-benzothiazepines

Benzothiazepines 1-3 inhibited acetylcholinesterase (AChE; EC 3.1.1.7) enzyme in a concentration-dependent fashion with IC(50) values of 1.0 +/- 0.002, 1.2 +/- 0.005 and 1.3 +/- 0.001 microM, respectively. By using linear-regression equations, Lineweaver-Burk, Dixon plots and their secondary replots were constructed which indicated that compounds 1-3 are non-competitive inhibitors of AChE with K(i) values of 0.8 +/- 0.04, 1.1 +/- 0.002, and 1.5 +/- 0.001 microM, respectively. Molecular docking studies revealed that all the compounds are completely buried inside the aromatic gorge of AChE, extending deep into the gorge of AChE. A comparison of the docking results of compounds 1-3 displayed that these compounds generally adopt the same binding mode in the active site of AChE. The superposition of the docked structures demonstrated that the non-flexible benzothiazepine always penetrate into the aromatic gorge through the six-membered ring A, which allowed the ligands to interact simultaneously with more than one subsites of the active center of AChE. The higher AChE inhibitory potential of compounds 1-3 was found to be the cumulative effect of hydrophobic contacts and pi-pi interactions between the ligands and AChE. The relatively high affinity of benzothiazepine 1 with AChE was found to be due to additional hydrogen bond in benzothiazepine 1-AChE complex. The results indicated that substitution of halogen and methyl groups by hydrogen at aromatic ring of the benzothiazepine decreased the affinity of these molecules towards enzyme that may be due to the polar non-polar repulsions of these moieties with the amino acid residues in the active site of AChE. The observed binding modes of benzothiazepines 1-3 in the active site of AChE explain the affinities of benzothiazepines and provide a rational basis for the structure-based drug design of benzothiazepines with improved pharmacological properties.     

Molecular dynamics simulation of Axillaridine–A: A potent natural cholinesterase inhibitor

Molecular Dynamics (MD) simulations were carried out for human acetylcholinesterase (hAChE) and its complex with Axillaridine–A, in order to dynamically explore the active site of the protein and the behaviour of the ligand at the peripheral binding site. Simulation of the enzyme alone showed that the active site of AChE is located at the bottom of a deep and narrow cavity whose surface is lined with rings of aromatic residues while Tyr72 is almost perpendicular to the Trp286, which is responsible for stable π -π interactions. The complexation of AChE with Axillaridine-A, results in the reduction of gorge size due to interaction between the ligand and the active site residues. The gorge size was determined by the distance between the center of mass of Glu81 and Trp286. As far as the geometry of the active site is concerned, the presence of ligand in the active site alters its specific conformation, as revealed by stable hydrogen bondings established between amino acids. With the increasing interaction between ligand and the active amino acids, size of the active site of the complex decreases with respect to time. Axillaridine-A, forms stable π -π interactions with the aromatic ring of Tyr124 that results in inhibition of catalytic activity of the enzyme. This π -π interaction keeps the substrate stable at the edge of the catalytic gorge by inhibiting its catalytic activity. The MD results clearly provide an explanation for the binding pattern of bulky steroidal alkaloids at the active site of AChE.     

Molecular docking study on the “back door” hypothesis for product clearance in acetylcholinesterase

Acetylcholinesterase (AChE) is one of the fastest enzymes known, even though the active site is buried inside the protein at the end of a 20-A deep narrow gorge. Among the great variety of crystal structures of this enzyme, both in the absence and presence of various ligands and proteins, the structure of a complex of AChE with the pseudo-irreversible inhibitor Mf268 is of particular interest, as it assists in the proposal of a back door for product clearance from the active site. Binding of Mf268 to AChE results in the carbamoylation of Ser200 and liberation of an eseroline-fragment as the leaving group. The crystal structure of the AChE-Mf268 complex, however, proves that eseroline has escaped from the enzyme, despite the fact that the Ser-bound inhibitor fragment blocks the gorge entrance. The existence of alternative routes other than through the gorge for product clearance has been postulated but is still controversially discussed in the literature, as an experimental proof for such a back door is still missing. We have used Monte Carlo-based molecular docking methods in order to examine possible alternative pathways that could allow eseroline to be released from the protein after being cleaved from the substrate by Ser200. Based on our results, a short channel at the bottom of the gorge seems to be the most probable back-door site, which begins at amino acid Trp84 and ends at the enzyme surface in a cavity close to amino acid Glu445. [Figure: see text].     

Kinetics and molecular docking studies of kaempferol and its prenylated derivatives as aldose reductase inhibitors

Aldose reductase inhibitors (ARIs) suppressing the hyperglycemia-induced polyol pathway have been provided as potential therapeutic candidates in the treatment and prevention of diabetic complications. Based upon structure-activity relationships of desmethylanhydroicaritin (1) and sophoflavescenol (2) as promising ARIs, 3,4'-dihydroxy flavonols with a prenyl or lavandulyl group at the C-8 position and a hydroxyl or methoxy group at the C-5 position are important for aldose reductase (AR) inhibition. In order to prove the above results, a combination of computational prediction and enzyme kinetics has begun to emerge as an effective screening technique for the potential. In the present study, we predicted the 3D structure of AR in rat and human using a docking algorithm to simulate binding between AR and prenylated flavonoids (1 and 2) and kaempferol (3) and scrutinized the reversible inhibition of AR by these ARIs. Docking simulation results of 1-3 demonstrated negative binding energies (Autodock 4.0=-9.11 to -7.64 kcal/mol; Fred 2.0=-79.54 to -51.84 kcal/mol) and an additional hydrogen bond through Phe122 and Trp219, in addition to the previously proposed interaction of AR and phenolics through Trp20, Tyr48, His110, and Trp111 residues, indicating that the presence of 8-prenyl and 5-methyl groups might potentiate tighter binding to the active site of the enzyme and more effective AR inhibitors. Moreover, types of AR inhibition were different depending on the presence or absence of the 8-prenyl group, in that 1 and 2 are mixed inhibitors with respective Ki values of 0.69 μM and 0.94 μM, while 3 showed noncompetitive inhibition with a Ki value of 4.65 μM. The present study suggests that an effective strategy for screening potential ARIs could be established by predicting 3D structural conformation of prenylated flavonoids and the orientation within the enzyme as well as by simultaneously determining the mode of enzyme inhibition.     

Structural insight into the inhibition of acetylcholinesterase by 2,3,4, 5-tetrahydro-1, 5-benzothiazepines

Benzothiazepines 1–3 inhibited acetylcholinesterase (AChE; EC 3.1.1.7) enzyme in a concentration-dependent fashion with IC₅₀ values of 1.0 ± 0.002, 1.2 ± 0.005 and 1.3 ± 0.001 μM, respectively. By using linear-regression equations, Lineweaver-Burk, Dixon plots and their secondary replots were constructed which indicated that compounds 1–3 are non-competitive inhibitors of AChE with K_i values of 0.8 ± 0.04, 1.1 ± 0.002, and 1.5 ± 0.001 μM, respectively. Molecular docking studies revealed that all the compounds are completely buried inside the aromatic gorge of AChE, extending deep into the gorge of AChE. A comparison of the docking results of compounds 1–3 displayed that these compounds generally adopt the same binding mode in the active site of AChE. The superposition of the docked structures demonstrated that the non-flexible benzothiazepine always penetrate into the aromatic gorge through the six-membered ring A, which allowed the ligands to interact simultaneously with more than one subsites of the active center of AChE. The higher AChE inhibitory potential of compounds 1–3 was found to be the cumulative effect of hydrophobic contacts and π-π interactions between the ligands and AChE. The relatively high affinity of benzothiazepine 1 with AChE was found to be due to additional hydrogen bond in benzothiazepine 1-AChE complex. The results indicated that substitution of halogen and methyl groups by hydrogen at aromatic ring of the benzothiazepine decreased the affinity of these molecules towards enzyme that may be due to the polar non-polar repulsions of these moieties with the amino acid residues in the active site of AChE. The observed binding modes of benzothiazepines 1–3 in the active site of AChE explain the affinities of benzothiazepines and provide a rational basis for the structure-based drug design of benzothiazepines with improved pharmacological properties.     

Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties

The withanolides 1-3 and 4-5 isolated from Ajuga bracteosa and Withania somnifera, respectively, inhibited acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes in a concentration-dependent fashion with IC50 values ranging between 20.5 and 49,2 microm and 29.0 and 85.2 microm for AChE and BChE, respectively. Lineweaver-Burk as well as Dixon plots and their secondary replots indicated that compounds 1, 3, and 5 are the linear mixed-type inhibitors of AChE, while 2 and 4 are non-competitive inhibitors of AChE with K(i) values ranging between 20.0 and 45.0 microm. All compounds were found to be non-competitive inhibitors of BChE with K(i) values ranging between 27.7 and 90.6 microm. Molecular docking study revealed that all the ligands are completely buried inside the aromatic gorge of AChE, while compounds 1, 3, and 5 extend up to the catalytic triad. A comparison of the docking results showed that all ligands generally adopt the same binding mode and lie parallel to the surface of the gorge. The superposition of the docked structures demonstrated that the non-flexible skeleton of the ligands always penetrates the aromatic gorge through the six-membered ring A, allowing their simultaneous interaction with more than one subsite of the active center. The affinity of ligands with AChE was found to be the cumulative effects of number of hydrophobic contacts and hydrogen bonding. Furthermore, all compounds also displayed dose-dependent (0.005-1.0 mg/mL) spasmolytic and Ca2+ antagonistic potentials in isolated rabbit jejunum preparations, compound 4 being the most active with an ED50 value of 0.09 +/- 0.001 mg/mL and 0.22 +/- 0.01 microg/mL on spontaneous and K+ -induced contractions, respectively. The cholinesterase inhibitory potential along with calcium antagonistic ability and safe profile in human neutrophil viability assay could make compounds 1-5 possible drug candidates for further study to treat Alzheimer's disease and associated problems.     

Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P(2) and Trp in P(2)('), consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P(2) and P(2)(') provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P(3), and P(3)(') amino acids identified an inhibitor with Trp in P(2), P(2)('), and P(3), and Phe in P(3)(') that bound to cathepsin P with a K(i) of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.     

Expression of tryptophan decarboxylase and tyrosine decarboxylase genes in tobacco results in altered biochemical and physiological phenotypes

The substrate specificity of tryptophan (Trp) decarboxylase (TDC) for Trp and tyrosine (Tyr) decarboxylase (TYDC) for Tyr was used to modify the in vivo pools of these amino acids in transgenic tobacco. Expression of TDC and TYDC was shown to deplete the levels of Trp and Tyr, respectively, during seedling development. The creation of artificial metabolic sinks for Trp and Tyr also drastically affected the levels of phenylalanine, as well as those of the non-aromatic amino acids methionine, valine, and leucine. Transgenic seedlings also displayed a root-curling phenotype that directly correlated with the depletion of the Trp pool. Non-transformed control seedlings could be induced to display this phenotype after treatment with inhibitors of auxin translocation such as 2,3,5-triiodobenzoic acid or N-1-naphthylphthalamic acid. The depletion of aromatic amino acids was also correlated with increases in the activities of the shikimate and phenylpropanoid pathways in older, light-treated transgenic seedlings expressing TDC, TYDC, or both. These results provide in vivo confirmation that aromatic amino acids exert regulatory feedback control over carbon flux through the shikimate pathway, as well as affecting pathways outside of aromatic amino acid biosynthesis.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP 1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D) 1H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs epsilon-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His3, Trp72, Tyr41, Tyr50 and Tyr74) appear to be exposed and accessible to 3-N-carboxymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp25 and Trp62 indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp72 ring in the presence of BASA. Moreover, a number of H beta resonances can be identified and sorted according to specific types of amino acid residues.     

Kinetics and structure-activity relationship studies on pregnane-type steroidal alkaloids that inhibit cholinesterases

The mechanism of inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes by 23 pregnane-type alkaloids isolated from the Sarcococca saligna was investigated. Lineweaver-Burk and Dixon plots and their secondary replots showed that the majority of these compounds, that is 1, 4, 5, 6, 9, 10, 12, 13, 15-19, and 21 were found to be noncompetitive inhibitors of both enzymes. Compounds 8, 20, 22, and 23 were determined to be uncompetitive inhibitors of BChE, while compounds 11 and 14 were found to be uncompetitive and linear mixed inhibitors of AChE, respectively. Ki values were found to be in the range of 2.65-250.0 microM against AChE and 1.63-30.0 microM against BChE. The structure-activity relationship (SAR) studies suggested that the major interaction of the enzyme-inhibitor complexes are due to hydrophobic and cation-pi interactions inside the aromatic gorge of these cholinesterases. The effects of various substituents on the activity of these compounds are also discussed in details.     

Three-dimensional structure of a complex of E2020 with acetylcholinesterase from Torpedo californica

The 3D structure of a complex of the anti-Alzheimer drug, E2020, also known as Aricep^®, with Torpedo californica acetylcholinesterase is reported. The X-ray structure, at 2.5 Å resolution, shows that the elongated E2020 molecule spans the entire length of the active-site gorge of the enzyme. It thus interacts with both the ‘anionic’ subsite, at the bottom of the gorge, and with the peripheral anionic site, near its entrance, via aromatic stacking interactions with conserved aromatic residues. It does not interact directly with either the catalytic triad or with the ‘oxyanion hole’. Although E2020 is a chiral molecule, and both the S and R enantiomers have similar affinity for the enzyme, only the R enantiomer is bound within the active-site gorge when the racemate is soaked into the crystal. The selectivity of E2020 for acetylcholinesterase, relative to butyrylcholinesterase, can be ascribed primarily to its interactions with Trp279 and Phe330, which are absent in the latter.     

Kringle 4 from human plasminogen: a proton magnetic resonance study via two-dimensional photochemically induced dynamic nuclear polarization spectroscopy

Two-dimensional (2D) proton magnetic resonance techniques used in conjunction with laser photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy have been applied to studying the kringle 4 domain from human plasminogen at 360 MHz. Out of 11 potential CIDNP-sensitive aromatic side chains, only 5 (His3, Tyr41, Tyr50, Trp72, and Tyr74) appear to be accessible to 3-(carboxymethyl)lumiflavin, the dye used to photogenerate spin polarization. Of these, Trp72 and Tyr74 are known to be at, or near, the lysine-binding site. The spin-spin scalar (J) and phase-sensitive dipolar (Overhauser) connectivities in the 2D experiments yield absolute assignments for the aromatic signals stemming from the exposed tyrosyl and tryptophanyl rings. Moreover, a number of side-chain H beta resonances can be identified and assigned to specific types of aromatic amino acid residues.     

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC₅₀ 216 μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC₅₀ values of 3.5 and 44.2 μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.     

Virtual screening and QSAR study of some pyrrolidine derivatives as α-mannosidase inhibitors for binding feature analysis

Virtual screening and QSAR analysis were carried out to investigate the binding features of (2R, 3R, 4S)-2-aminomethylpyrrolidine 3,4-diol and the functionalized pyrrolidine derivatives to the α-mannosidase I and II enzymes. The QSAR models (possessed considerable R², Q² values, etc.) suggested that the presence of polar property on the vdW surface (vsurf_W, vsurf_Wp, etc.) of the molecules is important along with the presence of aromatic rings (opr_violation) in the molecules (which also provide hydrophobicity to the molecules). The docking study performed on α-mannosidase I and II enzymes pointed that the main interactions occur by hydrogen bonds, hydrophobic π–π stacking contacts and salt bridges with the cation calcium (for α-mannosidase I) and close interaction with zinc ion (α-mannosidase II), respectively. The bond flexibility orientates the aromatic ring in the molecules toward the hydrophobic cavity for π–π stacking contacts with the aromatic amino acids (Phe528, Phe329 and Phe659 for α-mannosidase I and Trp95, Tyr269, Phe312, Tyr102 for α-mannosidase II). The pharmacophore analysis also supports the results derived from the docking and QSAR studies. Our results suggest that the best compound to inhibit both classes of α-mannosidase is the compound 30, which may be used to design similar and better inhibitors to next generation drugs.     

Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. alpestre

The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. ¹H and ¹³C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.     

Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.     

Role of amino acid hydrophobicity, aromaticity, and molecular volume on IAPP(20-29) amyloid self-assembly

Aromatic amino acids strongly promote cross-β amyloid formation; whether the amyloidogenicity of aromatic residues is due to high hydrophobicity and β-sheet propensity or formation of stabilizing π-π interactions has been debated. To clarify the role of aromatic residues on amyloid formation, the islet amyloid polypeptide 20-29 fragment [IAPP(20-29)], which contains a single aromatic residue (Phe 23), was adopted as a model. The side chain of residue 23 does not self-associate in cross-β fibrils of IAPP(20-29) (Nielsen et al., Angew Chem Int Ed 2009;48:2118-2121), allowing investigation of the amyloidogenicity of aromatic amino acids in a context where direct π-π interactions do not occur. We prepared variants of IAPP(20-29) in which Tyr, Leu, Phe, pentafluorophenylalanine (F5-Phe), Trp, cyclohexylalanine (Cha), α-naphthylalanine (1-Nap), or β-naphthylalanine (2-Nap) (in order of increasing peptide hydrophobicity) were incorporated at position 23 (SNNXGAILSS-NH2), and the kinetic and thermodynamic effects of these mutations on cross-β self-assembly were assessed. The Tyr, Leu, and Trp 23 variants failed to readily self-assemble at concentrations up to 1.5 mM, while the Cha 23 mutant fibrillized with attenuated kinetics and similar thermodynamic stability relative to the wild-type Phe 23 peptide. Conversely, the F5-Phe, 1-Nap, and 2-Nap 23 variants self-assembled at enhanced rates, forming fibrils with greater thermodynamic stability than the wild-type peptide. These results indicate that the high amyloidogenicity of aromatic amino acids is a function of hydrophobicity, β-sheet propensity, and planar geometry and not the ability to form stabilizing or directing π-π bonds.     

Amino acid residues in Rag1 crucial for DNA hairpin formation

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.     

Variation of amino acid properties in all-beta globular and outer membrane protein structures

Discriminating outer membrane (OM) proteins from globular proteins is an important task. The structural analysis of β-strands dominating globular (all-β) proteins and OM proteins provides useful insight to distinguish between them. In this work, we analyze the characteristic features of the 20 amino acid residues in all-β and OM proteins. We set up numerical indices for several properties of amino acid residues, such as, conformational parameters, surrounding hydrophobicity, accessible surface area and reduction in accessibility, and inter-residue contacts. We found that all the aromatic residues prefer to be in β-strands of both globular and OM proteins. The surrounding hydrophobicity of aromatic and non-polar amino acid residues in globular proteins is significantly higher than that of OM proteins. The residues Trp, Arg, Phe and Gln show a remarkable difference of reduction in accessibility between all-β globular (βG) and OM proteins. The positively charged residues, Lys and Arg in the membrane part of OM proteins have more number of contacts than globular proteins. Further, the behavior of the 20 amino acid residues in β-strand segments of globular and OM proteins have been discussed. The parameters developed in this work can be used for identifying transmembrane β-strands in OM proteins and for discriminating βG proteins from OM proteins.