[Show abstract][Hide abstract] ABSTRACT: The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.
[Show abstract][Hide abstract] ABSTRACT: Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called "aging", which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonaged adduct; a stereoselective preference for binding of the P(S)C(S) isomer of soman and the P(S) isomer of sarin was also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate, soman, and sarin. The P(S) stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The P(S) stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions.
[Show abstract][Hide abstract] ABSTRACT: The plasma form of the human enzyme platelet activating factor acetylhydrolase (PAF-AH) has been crystallized, and X-ray diffraction data were collected at a synchrotron source to a resolution of 1.47 A. The crystals belong to space group C2, with unit cell parameters of a = 116.18, b = 83.06, c = 96.71 A, and beta= 115.09 degrees and two molecules in the asymmetric unit. PAF-AH functions as a general anti-inflammatory scavenger by reducing the levels of the signaling molecule PAF. Additionally, the LDL bound enzyme has been linked to atherosclerosis due to its hydrolytic activities of pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids.
Protein and Peptide Letters 02/2009; 16(1):97-100. DOI:10.2174/092986609787049321 · 1.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5 angstroms. It has a classic lipase alpha/beta-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.
[Show abstract][Hide abstract] ABSTRACT: Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.
Journal of Proteome Research 06/2003; 2(3):255-63. DOI:10.1021/pr025584d · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The quantification of the packing of residues in proteins and docking of ligands to macromolecules is important in understanding
protein stability and drug design. The number of atoms in contact (within a distance of 4.5 Å) can be used to describe the
local environment of a residue. As this number increases, the accessible surface area (ASA) of the residue decreases exponentially
and the variation can be described in terms of an exponential equation of the form y = a1exp(–x/a2), each residue having its own set of parameters a1 and a2, which also depend on whether the whole residue or just the side chain is considered. Hydrophobic and hydrophilic residues
can be distinguished on the basis of both the average number of surrounding atoms and the variation of ASA. For a given number
of partner atoms, a comparison of the observed ASA with the expected value obtained from the equation provides a method of
assessing the goodness of packing of the residue in a protein structure or its importance in the binding of a ligand. The
equation provides a method to estimate the ASA of a protein molecule and the average relative accessibilities of different
residues, the latter being inversely correlated with hydrophobicity values.
Protein engineering 09/2002; 15(8):659-67. DOI:10.1093/protein/15.8.659
[Show abstract][Hide abstract] ABSTRACT: To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in alpha-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H...pi and N-H...pi) which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An alpha-helix and a contiguous 3(10)-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side.
[Show abstract][Hide abstract] ABSTRACT: To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in α-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H···π and N-H···π which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An α-helix and a contiguous 310-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side.
Protein Engineering Design and Selection 02/2002; 15(2):91-100. DOI:10.1093/protein/15.2.91 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Silver(I) oxide–silver halide mediated alcoholyses of racemic 2,4-di-O-benzoyl-myo-inositol 1,3,5-orthoformate, and its 6-O-methyl and 6-O-sulfonylated derivatives, under identical conditions have been compared. While only the 4-O-benzoyl group undergoes solvolysis in the former two, to yield the corresponding 2-O-benzoyl-myo-inositol 1,3,5-orthoformate, both the 4-O- as well as the 2-O-benzoyl groups undergo solvolysis in the latter, to yield racemic 6-O-sulfonyl-myo-inositol 1,3,5-orthoformates. These results show that solvolysis of the 2-O-benzoyl group in sulfonates is a consequence of intramolecular assistance by the sulfonyl group. Catalytic efficiency of the silver halides in bringing about solvolysis of the benzoates decreased in the order AgI > AgBr > AgCl. A reaction mechanism involving silver–inositol derivative chelates has been
Journal of the Chemical Society Perkin Transactions 2 01/2002; DOI:10.1039/b101476p
[Show abstract][Hide abstract] ABSTRACT: Instead of looking at the interfacial area as a measure of the extent of a protein--protein recognition site, a new procedure has been developed to identify the importance of a specific residue, namely tryptophan, in the binding process. Trp residues which contribute more towards the free energy of binding have their accessible surface area reduced, on complex formation, for both the main-chain and side-chain atoms, whereas for the less important residues the reduction is restricted only to the aromatic ring of the side chain. The two categories of residues are also distinguished by the presence or absence of hydrogen bonds involving the Trp residue in the complex. A comparison of the observed change in the accessible surface area with the value calculated using an analytical expression provides another way of characterizing the Trp residues critical for binding and this has been used to identify such residues involved in binding non-proteinaceous molecules in protein structures.
Protein engineering 02/2001; 14(1):7-15. DOI:10.1093/protein/14.1.7
[Show abstract][Hide abstract] ABSTRACT: Crystallography and proton NMR spectroscopy were used to compare the conformations of aryl amino Fischer carbene complexes with structurally analogous aryl carboxamides. The similarity disappears when the aromatic rings were complexed with tricarbonylchromium groups. Details of synthesis, spectral and analytical data for all new compounds are provided.
[Show abstract][Hide abstract] ABSTRACT: Although relatively rare, the tryptophan residue (Trp), with its large hydrophobic surface, has a unique role in the folded structure and the binding site of many proteins, and its fluorescence properties make it very useful in studying the structures and dynamics of protein molecules in solution. An analysis has been made of its environment and the geometry of its interaction with neighbors using 719 Trp residues in 180 different protein structures. The distribution of the number of partners interacting with the Trp aromatic ring shows a peak at 6 (considering protein residues only) and 8 (including water and substrate molecules also). The means of the solvent-accessible surface areas of the ring show an exponential decrease with the increase in the number of partners; this relationship can be used to assess the efficiency of packing of residues around Trp. Various residues exhibit different propensities of binding the Trp side chain. The aromatic residues, Met and Pro have high values, whereas the smaller and polar-chain residues have weaker propensities. Most of the interactions are with residues far away in sequence, indicating the importance of Trp in stabilizing the tertiary structure. Of all the ring atoms NE1 shows the highest number of interactions, both along the edge (hydrogen bonding) as well as along the face. Various weak but specific interactions, engendering stability to the protein structure, have been identified.
Proteins Structure Function and Bioinformatics 03/2000; 38(3):288-300. DOI:10.1002/(SICI)1097-0134(20000215)38:33.3.CO;2-Z · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The geometry of the interaction of the aromatic side chains of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and histidine (His) with the indole ring of Trp has been analyzed using the structures in the Protein Data Bank in order to understand the dependence of the packing behaviour on the size and chemical nature of the aromatic rings. The Phe ring prefers to interact either perpendicularly, with its edge pointing towards the Trp face, or in an offset-stacked arrangement. The edge-to-face motif is typical of a Trp-Trp pair. While parallel stacking is the dominant feature of Trp-His interaction, Tyr packs in a more uniform manner around Trp with a higher than expected occurrence at the edge and a few cases of possible OH-pi interaction.
[Show abstract][Hide abstract] ABSTRACT: The X-ray crystal structure of 2,3-dihydro-2,2,4-trimethyl-1H-1,5- benzodiazepine (L), which has medicinal effect on the central nervous system, has been determined. The solid state conformation of the seven-membered heterocyclic ring in L is found to be somewhat like an armchair. Its modes of binding to three metal ions Ni2+, Cu2+ and Zn2+ are identified. In cases of Ni2+ and Cu2+, the seven-membered heterocyclic ring in L opens up following a hydrolysis of the imino bond promoted by these two metal ions. Zn2+ cannot effect such ring opening.
[Show abstract][Hide abstract] ABSTRACT: The possibility of the π-face of a heterocyclic ring acting as a hydrogen-bond acceptor has considerable significance in the structure and binding of cofactors and nucleic acids to proteins. This interaction has been modeled using ab initio calculations on various complexes of pyridine with water, ammonia, methane, and benzene. Both Hartree−Fock (HF) and MP2/6-31G(d,p) calculations, including counterpoise corrections, have been carried out on a number of representative geometries. In addition to the expected hydrogen-bonded structure involving the nitrogen lone pair, a number of other orientations in which X−H is placed above the π-face are also found to be energetically favorable. The maximum stabilization is found directly above the pyridine nitrogen for water and ammonia, whereas for methane it is shifted to a point halfway toward the ring center. The corresponding complexation energies are 2.9 (X = O), 1.8 (N), and 0.8 (C) kcal mol-1, which are 0.45, 0.56, and 0.71, respectively, of the values obtained when the interaction is in the conventional hydrogen-bonded geometry. Bifurcated structures, with the XH2 group above the pyridine ring but displaced from the center toward the nitrogen, are also found to be fairly stabilized. A herringbone structure with two of the benzene C−H bonds facing the pyridine ring is computed to have a stabilization energy of 2.7 kcal mol-1, which is greater by 0.4 kcal mol-1 than that for the linear C−H···N hydrogen-bonded geometry involving the nitrogen lone pair. The interaction energies with the π-face are of comparable magnitude for benzene and pyridine. The computed relative energetics for various geometries should be useful in developing potential functions for modeling the binding of cofactors and nucleic acids with proteins.
The Journal of Physical Chemistry A 10/1998; 102(45). DOI:10.1021/jp981501y · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protected myo-inositol derivatives are important precursors in the synthesis of phosphorylated myo-inositol derivatives, which play a significant role in cellular signal transduction. The structure of the title compound, C14H14O7, which was prepared from myo-inositol, has been determined by X-ray crystallography. Several types of hydrogen-bonding interactions are involved in the packing of the molecule in the crystal.
[Show abstract][Hide abstract] ABSTRACT: (±)-2,4-Di-O-benzoyl-myo-inositol 1,3,5-orthoformate, on heating in the presence of a base, undergoes transesterification to give 2,4,6-tri-O-benzoyl-myo-inositol 1,3,5-orthoformate and 2-O-benzoyl-myo-inositol 1,3,5-orthoformate in the solid state. The same reaction can also be performed by microwave irradiation instead of heating. The crystal structure of the dibenzoate reveals that the screw-axis-related molecules have the hydroxyl and the carbonyl groups ideally oriented for the reaction and gives a close picture of how such a reaction proceeds in enzymes. The structure of the corresponding acetate, (±)-2-O-benzoyl-4-O-acetyl-myo-inositol 1,3,5-orthoformate, lacks this geometry and hence is unreactive in the solid state. Both the acetate and the benzoate undergo base-catalyzed transesterification in solution.
Journal of the American Chemical Society 04/1998; 120(16). DOI:10.1021/ja9731332 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2,3-Dihydro-3-hydroxybenzisothiazole 1,1-dioxides constitute a self-complementary hydrogen-bond donor−acceptor system. 2,3-Dihydro-2-isopropyl-3-hydroxybenzisothiazole 1,1-dioxide exhibits interesting concentration and temperature-dependent NMR spectra due to association in solution. The intermolecular association of these molecules through S−O···H−O interactions resulting in the formation of novel hydrogen-bonded dimers containing 12/14-membered bicyclic bridge is revealed by X-ray crystallography. X-ray crystallographic studies also confirm the existence of N-alkyl-2-formylbenzenesulfonamides exclusively as 2,3-dihydro-2-alkyl-3-hydroxybenzisothiazole 1,1-dioxides in the solid state.
The Journal of Organic Chemistry 01/1998; 63(2):230-234. DOI:10.1021/jo962352n · 4.72 Impact Factor