Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site

The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.     

Molecular modeling studies on the interactions of aflatoxin B1 and its metabolites with the peripheral anionic site of human acetylcholinesterase

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them, aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic for mammals. It can contaminate many kinds of food like seeds, oil, olives, milk, dairy products, corn and meat, causing acute and chronic damages to the organism, especially in the liver, being, for this reason, considered highly hepatotoxic. AFB1 is also a mixed inhibitor of the enzyme acetylcholinesterase (AChE). This fact, together with its high toxicity and carcinogenicity, turns AFB1 into a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited molecular modeling studies on the interactions of AFB1 and its metabolites inside the peripheral anionic site of human AChE (HssAChE), to verify their stability, suggest the preferential ways of inhibition, and compare their behavior to each other. Our results suggest that all metabolites can be better inhibitors of HssAChE than AFB1 and that AFBO and AFM1, the most toxic and carcinogenic metabolites of AFB1, are also the most effective HssAChE inhibitors among the AFB1 metabolites.

Communicated by Ramaswamy H. Sarma     

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.     

A hydrophobic pocket in the active site of glycolytic aldolase mediates interactions with Wiskott-Aldrich syndrome protein

Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, four-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds around the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C terminus with its active site and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure, a novel naphthol phosphate-based inhibitor of aldolase was identified, and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.     

The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues

The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His(308) by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate.     

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure-activity relationships for inhibitions by aryl carbamates

Substituted phenyl-N-butyl carbamates (1-9) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme inhibitor adduct (Ki), the inactivation constant of the enzyme-inhibitor adduct (k2), and the bimolecular inhibition constant (k(i)) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1-9 and the Hammett substituent constant (sigma), are observed, and the reaction constants (ps) are -1.36, 0.35 and -1.01, respectively. Therefore, the above reaction may form a positive charged enzyme-inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.     

Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1

We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.     

The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site.

The cleavage step of bacterial RNase P catalysis involves concentration-independent processes after the formation of the ribozyme-substrate complex that result in the breaking of a phosphodiester bond. The 2'OH group at the cleavage site of a pre-tRNA substrate is an important determinant in the cleavage step. We determined here that in contrast to a tRNA substrate, the 2'OH at the cleavage site of two in vitro selected substrates has no effect, whereas a 2'OH located adjacent to the cleavage site has a similarly large effect on the cleavage step. This result indicates that a unique 2'OH in the vicinity of the cleavage site interacts with the ribozyme to achieve the maximal efficiency of the cleavage step. Individual modifications in a pre-tRNA substrate that disrupt ES interactions proximal to the cleavage site generally have little effect on the usage of this unique 2'OH. Ribozyme modifications that delete the interactions involving the T stem-loop of the tRNA have a large effect on the usage of this unique 2'OH and also alter the location of this 2'OH. We propose a new ES complex prior to the bond-breaking step in the reaction scheme to explain these results. This second ES complex is in fast equilibrium with the initial ES complex formed by bimolecular collision. The ribozyme interaction with this unique 2'OH shifts the equilibrium in favor of the second ES complex. The formation of the second ES complex may require optimal geometry of the two independently folding domains of this ribozyme to precisely position crucial functional groups and Mg2+ ions in the active site. Such a domain geometry is significantly favored by the RNase P protein. In the absence of the protein, spatial rearrangement of these domains in the ES complex may be necessary.     

Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and acylation sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.     

Probing the peripheral anionic site of acetylcholinesterase with quantitative structure activity relationships for inhibition by biphenyl-4-acyoxylate-4'-N-Butylcarbamates

Biphenyl-4-acyoxylate-4'-N-butylcarbamates 1-8 are synthesized from 4,4'-biphenol and are characterized as the pseudosubstrate inhibitors of acetylcholinesterase. In other words, the inhibitors bind to the enzyme and react with the enzyme to form the tetrahedral intermediates for the K(i) steps, and then the tetrahedral intermediates exclude the leaving groups to form a common N-butycarbamyl enzyme intermediate for the k(c) steps. Due to a linear character of the 4,4'-biphenyl moiety, the 4'-N-butylcarbamate moieties of the inhibitors react with the Ser200 residue of the enzyme while the 4-acyoxylate moieties of the inhibitors, on the other hand, should fit in the peripheral anionic site of the enzyme, which is located at the mouth of the deep active site gorge. Thus, carbamates with varied acyl substituents at the 4-position of the biphenyl ring are good candidates for probing the quantitative structure activity relationships for the peripheral anionic site of the enzyme. The fact that the pK(i), log k(c), and log K(i) values are correlated with neither the Taft substituent constant (sigma*) nor the Taft steric constant (E(s)) indicates that the 4-acyoxylate moieties of the inhibitors are too far away from the reaction center. However, the pK(i), log k(c), and log K(i) values are linearly correlated with the Hansch hydrophobicity constant, pi. The intensity constants (psi) for these correlations are 0.16, -0.035, and 0.13, respectively. These results indicate that interactions between the 4-acyoxylate groups of the inhibitors and the peripheral anionic site of the enzyme are mainly hydrophobic ones. The correlation results are slightly improved by using the two-parameter correlations with the Taft substituent steric constant, E(s), and pi. For pK(i), log k(c), and log K(i)-E(s)-pi correlations, the psi values are 0.21, -0.021, and 0.19, respectively; the intensity constants for steric effect (delta) are 0.08, 0.022, and 0.10, respectively. Besides hydrophobic interactions, the two-parameter correlations also suggest that little steric hindrance occurs for the bulkier inhibitors to pass by the peripheral anionic site of the enzyme.     

Drug selectivity is determined by coupling across the NAD+ site of IMP dehydrogenase

Drug resistance often results from mutations that are located far from the drug-binding site. The effects of these mutations are perplexing. The inhibition of IMPDH by MPA is an example of this phenomenon. Mycophenolic acid (MPA) is a species-specific inhibitor of IMPDH; mammalian IMPDHs are very sensitive to MPA, while the microbial enzymes are resistant to the inhibitor. MPA traps the covalent intermediate E-XMP and binds in the nicotinamide half of the dinucleotide site. Previous results indicated that about half of the difference in sensitivity derives from residues in the MPA-binding site [Digits, J. A., and Hedstrom, L. (1999) Biochemistry 38, 15388-15397]. The remainder must be attributed to regions outside the MPA-binding site. The adenosine subsite of the NAD+ site is not conserved among IMPDHs and is, therefore, a likely candidate. Our goal is to examine the coupling between the nicotinamide and adenosine sites in order to test this hypothesis. We performed multiple inhibitor experiments with the Tritrichomonas foetus and human type 2 IMPDHs using tiazofurin and ADP, which bind in the nicotinamide and adenosine subsites, respectively. For T. foetus IMPDH, tiazofurin and ADP are extraordinarily synergistic. In contrast, these inhibitors are virtually independent for the human type 2 enzyme. We suggest that the difference in coupling of the nicotinamide and adenosine subsites accounts for the remaining difference in MPA affinity between T. foetus and human IMPDH.     

Trophic interactions of Antarctic seals as determined by stable isotope signatures

The diets and trophic interactions among Weddell, crabeater, Ross, and leopard seals in the eastern Ross Sea, Antarctica, were investigated by the use of stable isotope techniques during the 1999–2000 summer seasons. The ¹³C and ¹⁵N values in seal serum clearly distinguished the three Antarctic pack-ice seal species at different trophic positions (Weddell>Ross>crabeater). These patterns appeared to reflect a close linkage to their known foraging ecology and diving behaviors, and agreed well with their presumed dietary diversity. The more enriched ¹³C and ¹⁵N values in male Weddell seals than those in females suggested differences in foraging preferences between them. Significant differences in ¹⁵N were also found among different age groups of Weddell seals. A strong correlation between the C:N ratios and serum cholesterol was probably due to extremely high cholesterol levels in phocids. Comparisons of isotope data with harbor seals revealed distinct differences between Antarctic phocids and the northern seal species.     

The compact conformation of fibronectin is determined by intramolecular ionic interactions.

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.     

Probing the acyl binding site of acetylcholinesterase by protein engineering

Recombinant acetylcholinesterase from rat brain and two mutants were studied for their hydrolytic activity toward acetyl- and butyrylthiocholine substrates and for their sensitivity toward organophosphate and carbamate inhibitors. Both mutants, a point mutant where F295 was replaced by leucine, and a second mutant where loop PQES was replaced by SG, were designed for increased size of the acyl binding pocket. Wild type and mutant enzymes were expressed in baculovirus-infected insect cells and biochemically characterized. As expected, wild type rat brain acetylcholinesterase hydrolyzed acetylthiocholine, but not butyrylthiocholine. Sensitivity toward small- and medium-sized organophosphate inhibitors like paraoxon-methyl and paraoxon-ethyl was comparable, but bulky organophosphates like ethoprophos were less efficient inhibitors. This tendency applied to carbamates as well, since small carbamoyl moieties like carbofuran and aldicarb were stronger inhibitors than furathiocarb which features a bulky carbamoyl moiety. In contrast to wild type enzyme, both mutants were capable of hydrolyzing butyrylthiocholine. However, k_cat/K_m toward acetylthiocholine of the F295L mutant was reduced if compared to the wild type enzyme. All five organophosphate and three carbamate inhibitors inhibited mutant F295L more efficiently than the wild type enzyme.     

Peritonitis determined by the site of intra-abdominal surgery

The aim of this study was to determine bacterial flora infecting the peritoneal cavity during intraabdominal surgery by site of operation. Three groups of patients were examined. 29 patients who underwent surgery on the stomach, duodenum, biliary tract or pancreas, 15 patients operated on because of acute appendicitis and 63 patients operated on because of colon or rectum tumours. At the end of the operation but before closure cultures were obtained by swab from the completed anastomosis site. Samples were placed into transport medium and transported promptly to the laboratory. The results of the bacteriological examinations showed that the peritoneal cavity of all patients operated on were infected with bacteria characteristic for the digestive tract, especially by Enterobacteriaceae spp., Enterococcus spp. and Bacteroides spp. From patients operated on because of rectum or colon tumours 3 or 4 bacterial species were isolated most often and they were often infected with P. aeruginosa and C. albicans. This was in contrast to patients from the other groups. In patients infected with polymicrobial flora, B. fragilis and E. coli or enterococci and E. coli and enterococci were most often seen.     

The active site of hemerythrin as determined by X-ray absorption fine structure

Extensive X-ray absorption fine structure measurements and analysis have been made on azidomet- and methemerythrin and on the native forms of oxy- and deoxyhemerythrin. Due to the availability of models that have been synthesized to mimic the active site of hemerythrin, it was possible to make a thorough assessment of the various errors in the structural parameters determined by the analysis. It is found that the largest source of error is the lack of complete transferability of amplitude and phase between the standards and hemerythrin. This is of particular importance in distinguishing the contributions of the second-shell low-Z atoms and, thus, has a substantial influence on the determination of the iron-iron distance. The internal consistencies of the various checks and a new formulation of error analysis for the structural parameters give us confidence in the structure determined for the active site. The main result is that as O2 is released from oxyhemerythrin, the mu-oxo bridge between the two iron atoms in the active site with an Fe-O distance of 1.8 A converts to a mu-hydroxo bridge in deoxyhemerythrin, expanding the Fe-O distance to 2.0 A. The Fe-Fe distance expands proportionally from 3.24 A in oxyhemerythrin to 3.57 A in deoxyhemerythrin so as to keep the Fe-O-Fe bridging angle approximately constant. These conclusions provide experimental support for the structures of oxy- and deoxyhemerythrin proposed previously on the basis of spectroscopic and preliminary X-ray crystallographic data.     

The gross architecture of an antibody-combining site as determined by spin-label mapping

1. A series of Dnp (dinitrophenyl) nitroxide spin labels was used to map the dimensions of the combining site of the Dnp-binding immunoglobulin A myeloma protein MOPC 315. The method compares the observed e.s.r. (electron-spin-resonance) hyperfine splittings with those calculated on the basis of different postulated motions for the spin label. The analysis is complicated by the sensitivity of the e.s.r. hyperfine splitting to the overall ;tumbling' time of the antibody-hapten complex and the polarity of the spin-label's environment. When these effects are considered quantitatively, it is then possible to determine the degree of mobility of each hapten which is allowed by the shape of the combining site. 2. The dinitrophenyl ring is rigidly held, and the depth of the site is 1.1-1.2nm and has lateral dimensions at the entrance to the site >/=0.6nmx0.9nm. The analysis of the results for spin-labelled haptens with chiral centres allows these lateral dimensions to be refined to 0.8nm and 1.1nm, and it is shown that the site is asymmetric with respect to the plane of the dinitrophenyl ring. 3. A polarity profile of the combining site was also obtained and a positively charged amino acid residue, possibly arginine-95(L) (light chain), was located at the entrance to the site. 4. The binding of Gd(III) to the antibody-hapten complexes results in quenching of the e.s.r. signal of the nitroxide. By using La(III) as a control, the paramagnetic contribution to the quenching is measured. 5. Analysis of the differential quenchings of the enantiomers of two five-membered nitroxide ring spin labels gives two possible locations of the metal-binding site. One of these is equidistant (0.7nm) from each of the three dinitrophenyl aromatic protons, and nuclear-magnetic-resonance relaxation studies, at 270MHz, on solutions of dinitrobenzene, Gd(III) and the Fv fragment (variable region of heavy and light chain) from protein MOPC 315 support this location for the metal site. 6. The e.s.r. and metal-binding data were then compared with the results of a model of the combining site constructed on the basis of framework invariance in immunoglobulins [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol.41, in the press]. The overall agreement is very good. Assignments of possible chelating groups for the metal can be made.