Torpedo californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4D motif.

Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 , and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3.

The impact of crystallization conditions on structure-based drug design: A case study on the methylene blue/acetylcholinesterase complex.

Structure-based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ∼3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π-cation interactions with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design.

The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer methylene blue.

The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of (1)O(2) by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp(84) and Trp(279), present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of (1)O(2) on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase.

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ~8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen.

The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ((1)O(2)) trap, 9,10-dimethylanthracene, was retarded in the presence of AChE, and the rate of photo-inactivation was increased in the presence of D(2)O, indicating that inactivation was due to (1)O(2) generated by the photosensitizer. CD revealed slightly reduced far-UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo-oxidized AChE. However, both near-UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo-oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N'-formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo-oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross-linked dimers, whereas irradiation of a partially unfolded state generated higher-order oligomers. The active-site gorge of AChE contains Trp in inhibitor-binding sites that might be targets for photo-oxidation. Indeed, reversible inhibitors retard photo-inactivation, and photo-inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo-inactivation by sequestering the photosensitizer. Affinity photo-oxidation of AChE by MB thus provides a valuable model for studying site-specific photo-inactivation of enzymes in both fundamental and clinical contexts.

Evaluation of one-stage laparoscopic-assisted restorative proctocolectomy at a specialty center: comparison with the open approach.

PURPOSE: This study compared outcomes after laparoscopically assisted and open restorative proctocolectomy performed as a one-stage procedure, including anorectal mucosectomy and omission of ileal diversion. METHODS: We reviewed our prospectively maintained database of patients who underwent restorative proctocolectomy between 1998 and 2006. Demographic data, surgical indications, and intraoperative and postoperative complications were evaluated. Anastomotic leaks were identified by radiologic, endoscopic, or intraoperative evidence. The primary outcome variables were complications, duration of operation, blood loss, intraoperative spillage of enteric contents, and the ability to complete the procedure in one stage. RESULTS: One-stage laparoscopically assisted restorative proctocolectomy was performed in 50 patients and open restorative proctocolectomy was performed in 155 patients. The mean operative time was longer for the laparoscopically assisted group (198.7 vs. 159.1 minutes; P = 0.006). The mean estimated blood loss was less among the patients in the laparoscopically assisted group (287.5 vs. 386.4 ml; P = 0.006). There were no significant differences in intraoperative or postoperative complications between the two groups. CONCLUSIONS: Laparoscopically assisted one stage restorative proctocolectomy is a safe and technically feasible procedure. There seems to be no increase in the rate of postoperative complications compared with the open approach. Laparoscopically assisted restorative proctocolectomy should be considered in the surgical management of patients who require this procedure.

Recurrence patterns after first resection for stricturing or penetrating Crohn's disease.

BACKGROUND: Crohn's disease (CD) usually recurs after resection, but the factors associated with this risk remain obscure. We set out to determine the role of stricturing (Montreal Classification B2) versus penetrating (Classification B3) disease behavior in predicting early (<3 years) versus late (>or=3 years) postoperative recurrence. METHODS: We identified a cohort of 34 patients seen at The Mount Sinai Hospital who had undergone a first ileocolic resection prior to December 31, 2004, who had been clinically thought to have had stricturing (B2) disease, and for whom we could verify 1) the operative and surgical pathology findings; and 2) the time of onset of symptoms attributable to recurrent CD by endoscopy, radiology, or surgery. Cases were reclassified as either "stricturing" (B2) or "penetrating" (B3) on the basis of operative and surgical pathology reports. Recurrences were classified as either "early" (<3 years) or "late" (>or=3 years) depending on the first appearance of postoperative symptoms that were verified endoscopically and histologically, radiologically, or surgically as being attributable to anastomotic recurrence of the CD. RESULTS: Among these 34 patients clinically thought to have had B2 disease, 12 had B2 disease confirmed upon review of surgical and pathology reports and none of them had recurrence within 3 years. Among the 22 patients reclassified as B3 disease, 12 (55%) had early recurrence. This difference was significant at the 0.002 level by the Fisher Exact Test. CONCLUSIONS: There is a strong proclivity for early postoperative recurrence of penetrating CD compared to stricturing disease, which may not be evident by behavioral classification on clinical grounds alone. Patients with confirmed uncomplicated stricturing obstruction at their first resection seem unlikely to experience a clinical recurrence within the next 3 years.

Stabilization of Torpedo californica acetylcholinesterase by reversible inhibitors.

The dimeric form of Torpedo californica acetylcholinesterase provides a valuable experimental system for studying transitions between native, partially unfolded, and unfolded states since long-lived partially unfolded states can be generated by chemical modification of a nonconserved buried cysteine residue, Cys 231, by denaturing agents, by oxidative stress, and by thermal inactivation. Elucidation of the 3D structures of complexes of Torpedo californica acetylcholinesterase with a repertoire of reversible inhibitors permits their classification into three categories: (a) active-site directed inhibitors, which interact with the catalytic anionic subsite, at the bottom of the active-site gorge, such as edrophonium and tacrine; (b) peripheral anionic site inhibitors, which interact with a site at the entrance to the gorge, such as propidium and d-tubocurarine; and (c) elongated gorge-spanning inhibitors, which bridge the two sites, such as BW284c51 and decamethonium. The effects of these three categories of reversible inhibitors on the stability of Torpedo californica acetylcholinesterase were investigated using spectroscopic techniques and differential scanning calorimetry. Thermodynamic parameters obtained calorimetrically permitted quantitative comparison of the effects of the inhibitors on the enzyme's thermal stability. Peripheral site inhibitors had a relatively small effect, while gorge-spanning ligands and those binding at the catalytic anionic site, had a much larger stabilizing effect. The strongest effect was, however, observed with the polypeptide toxin, fasciculin II (FasII), even though, in terms of its binding site, it belongs to the category of peripheral site ligands. The stabilizing effect of the ligands binding at the anionic subsite of the active site, like that of the gorge-spanning ligands, may be ascribed to their capacity to stabilize the interaction between the two subdomains of the enzyme. The effect of fasciculin II may be ascribed to the large surface area of interaction (>2000 A(2)) between the two proteins. Stabilization of Torpedo californica acetylcholinesterase by both divalent cations and chemical chaperones was earlier shown to be due to a shift in equilibrium between the native state and a partially unfolded state ( Millard et al. ( 2003 ) Protein Sci. 12 , 2337 - 2347 ). The low molecular weight inhibitors used in the present study may act similarly and can thus be considered as pharmacological chaperones for stabilizing the fully folded native form of the enzyme.

Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones.

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t(1/2) approximately 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca(2+), Mg(2+), and Mn(2+) can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg(2+) can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39 degrees C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.