Whanchul Shin

Conformational flexibility in mammalian 15S‐lipoxygenase: Reinterpretation of the crystallographic data

Lipoxygenases (LOXs) are a family of nonheme iron dioxygenases that catalyze the regioselective and stereospecific hydroperoxidation of polyunsaturated fatty acids, and are involved in a variety of inflammatory diseases and cancers. The crystal structure of rabbit 15S‐LOX1 that was reported by Gillmor et al. in 1997 has played key roles for understanding the properties of mammalian LOXs. In this structure, three segments, including 12 residues in the superficial α2 helix, are absent and have usually been described as “disordered.” By reinterpreting the original crystallographic data we were able to elucidate two different conformations of the molecule, both having well ordered α2 helices. Surprisingly, one molecule contained an inhibitor and the other did not, thereby adopting a closed and an open form, respectively. They differed in the conformation of the segments that were absent in the original structure, which is highlighted by a 12 Å movement of α2. Consequently, they showed a difference in the size and shape of the substrate‐binding cavity. The new model should provide new insight into the catalytic mechanism involving induced conformational change of the binding pocket. It may also be helpful for the structure‐based design of LOX inhibitors. Proteins 2008.

Crystal structure of the C107S/C112S mutant of yeast nuclear 2‐Cys peroxiredoxin

Introduction. Peroxiredoxins (Prxs) are a superfamily of antioxidant enzymes, which are abundant in several isoforms in all kingdoms. Prxs catalyze the reduction of deleterious substances such as hydrogen peroxide (H2O2), alkyl hydroperoxides, and peroxynitrites by utilizing the thiol group of the “peroxidatic” cysteine (CP), which is conserved within the N-terminal region. Some eukaryotic Prxs also act as regulators of H2O2-mediated signal transduction. All Prxs belonging to the thioredoxin-fold superfamily share the same peroxidatic active-site structure. During a catalytic cycle, the CP residue is oxidized by peroxides to a cysteine sulfenic acid (CP-SOH) intermediate. Prxs are classified into 1-Cys and 2-Cys type based on the occurrence of the “resolving” Cys (CR) residue. The 1-Cys Prxs do not contain a CR residue, and the CP-SOH is recycled by glutathionylation mediated by glutathione S-transferase , followed by spontaneous reduction of the enzyme with glutathione. In 2-Cys Prxs, the CP-SOH and CR-SH react to form a stable disulfide, which is then reduced by oxidoreductases such as thioredoxin, tryparedoxin, AhpD, or AhpF. The 2-Cys Prxs have been further subdivided into “typical” and “atypical” types, depending on the position of the CR residue. In typical 2-Cys Prxs (hereafter referred to as T2-Cys Prxs), the CR residue is located within the C-terminal arm of another subunit of a homodimer. In contrast, the CR residue in an atypical 2-Cys Prx resides within the same subunit. The atypical 2-Cys Prxs have also been further subdivided into “L,” “C,” and “R” type subfamilies (hereafter referred to as L-, C-, and R2-Cys Prxs, respectively), depending on the spatial location of the CR residue. 6 Therefore, from a mechanistic point of view, there are five unique Prx subfamilies in total. To date, five distinct Prxs have been identified in the yeast Saccharomyces cerevisiae. They include three thiol peroxidases (cTPx I, II, and III) localized in the cytoplasm, one (nTPx) in the nucleus and one (mTPx) in the mitochondria. cTPx I, II, and III are T2-Cys Prxs, while mTPx is a 1-Cys Prx. nTPx is a member of the C2-Cys Prxs that contains a CxxxxC motif. Bacterial homologues of such Prxs are frequently referred to as the bacterioferritin comigratory proteins (BCP) and their plant homologues are named as PrxQ. These Prxs are least characterized among the Prx subfamilies and information regarding their structure is not yet available. In this study, we have determined the crystal structure of a truncated mutant of nTPx in which both the catalytic residues of Cys107 and Cys112 were replaced with serine. This mutant protein was gradually and spontaneously degraded by the freezing and thawing process until 56 amino acid residues were cleaved off from its N-terminal. nTPx has nuclear targeting sequences but the cleavage site has not yet been determined. The truncated mutant nTPx (hereafter referred to as tmTPx) may correspond to a physiologically mature nTPx. The present structure of a C2-Cys Prx makes it possible to compare the 3D structures of all the five Prx subfamilies.

An abdominal aortic aneurysm in an 8-month-old girl with tuberous sclerosis.

The association between an abdominal aortic aneurysm (AAA) and tuberous sclerosis (TS) is rare. An 8-month-old girl presented with a seizure, and the clinical evaluation revealed TS. An abdominal evaluation showed a 3-cm infrarenal AAA. A normal diameter of infrarenal aorta for an 8-month-old girl is about 6mm. The patient underwent an open repair with a polytetrafluoroethylene (PTFE) prosthesis. The pathology showed a loss of elastin fibres in the media of the aorta. The graft was patent on computed tomography (CT) angiography, performed 4 months after the operation. However, the patient died of complications related to seizures 5 years after the surgery. The graft remained patent until the time of death.

Computer modeling of the rhamnogalacturonase-"hairy" pectin complex.

The structure of a pectin‐bound complex of rhamnogalacturonase was modeled to identify the amino acid residues involved in catalysis and substrate binding. The “hairy” region of pectin, represented by six repeating stretches of (1→4)‐D‐galacturonate‐(1→2)‐L‐rhamnose dimer, was flexibly docked into the putative binding site of rhamnogalacturonase from Aspergillus aculeatus whose X‐ray structure is known. A search of the complex configurational space was performed using AutoDock for the dimeric and tetrameric sugar units in which the −1 galacturonate residue has various ring conformations. Then the plausible AutoDock solutions were manually extended to the dodecameric pectin models. Subsequently, the resulting complex models were subjected to solvated molecular dynamics using AMBER. In the best model, the substrate has an extended pseudo‐threefold helix with the −1 ring in a 4H3 half‐chair that approaches the transition state conformation. The catalytic machinery is clearly defined: Asp197 is a general acid and the activated water bound between Asp177 and Glu198 is a nucleophile. The active site is similar, with a small yet significant difference, to that of polygalacturonase that degrades the pectic “smooth” region of linear homopolymer of D‐(1→4)‐linked galacturonic acid. Rhamnogalacturonase has ten binding subsites ranging from −3 to +7, while polygalacturonase has eight subsites from −5 to +3. The model suggests that the eight amino acids including three arginine and three lysine residues, all of which are invariantly conserved in the rhamnogalacturonase family of proteins, are important in substrate binding. The present study may aid in designing mutational studies to characterize rhamnogalacturonase. Proteins 2004.

Novel receptor surface approach for 3D-QSAR: the weighted probe interaction energy method.

A 3D-QSAR technique, called the WeP (weighted probe interaction energy) method, has been developed based on the notion that certain regions of the receptor surface contribute, to varying extents, to the differences in the activities of the ligands, while other regions do not. The probes, placed around the surface of a superimposed set of ligands, were associated with fractional weights, and then an optimal distribution of probe weights that accounts for the activity profile of the training ligands was determined using a genetic algorithm. It has been shown for the three test samples that the pseudoreceptors, which consist of the surviving probes with nonzero weight values, have good predictabilities. Especially, in the case of dihydrofolate reductase inhibitors, the pseudoreceptor resembles the real protein in that there is no surviving probe in the solvent-exposed region.

A non-peptide angiotensin II receptor antagonist : 2-butyl-6-dimethoxymethyl-5-phenyl-1-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-imidazo[5,4-b]pyridine

The title compound, C33H33N7O2, is one of a series of imidazo[4,5-b]pyridine-based angiotensin II receptor antagonists showing high antihypertensive activity. The biphenyltetrazole moiety assumes the same conformation as in related compounds, but its relative orientation with respect to the central fused ring is different to that in these compounds, indicating that there is considerable conformational flexibility about the methylene bridge joining the two ring systems.

Flexible Alignment of Small Molecules Using the Penalty Method

An efficient flexible alignment method using the penalty method, called FAP, is described. FAP is a pairwise alignment algorithm that matches a flexible sample to a rigid template. It is a pure atom-based 3D method that utilizes the modified SEAL similarity index combined with an energy penalty term. The penalty term, defined as the third power of the ratio of the local strain energy to its target value, enables effective control of energy increase during alignment. The alignment procedure consists of the seed conformer generation, rigid-body alignment, and flexible optimization steps. Both conformation and alignment spaces are efficiently explored by the sparse, random sampling schemes. FAP has been tested with benchmark sets of seven different classes of ligands taken from the literature. In terms of the ability to produce the bioactive overlays, FAP is comparable to, or in some cases better than, other alignment methods. FAP is accurate, objective, fully automated, and fast enough to be used as a tool for virtual screening.

Structure of thiamin naphthalene-1,5-disulfonate monohydrate

Amino-2-methyl-5-pyrimidinio)- methyl)-5- (2-hydroxyethyl)-4-methylthiazolium naphthalene-l,5-disulfonate monohydrate, C~2 H~s- N4OS2÷.C 10H6(SO~)2.H20, M r = 570.65, ortho- rhombic, P212~2 ~, a = 7.887 (2), b = 15.754 (3), c

Flexible molecular superposition: development of a combined similarity index and application of the constrained optimization technique

I t is an important approach in drug design to correlate the three-dimensional (3D) structures of the drug molecules with their measures of biological activity, especially when the 3D structure of the receptor is not known.1 The 3D structures of a series of the drug molecules have to be mutually aligned to find a correlation, because only relative differences between many molecules can be related to variations in biological activity. Molecular superposition remains an important step in various design strategies such as the derivation of the pharmacophore model, the receptor mapping, the 3D quantitative structure-activity relationship (QSAR) analysis, and also the 3D structural database search. Numerous methods have been proposed for molecular superposition or structural alignment. These methods can be classified largely into pointbased or property-based methods. In point-based methods, pairs of atoms or pharmacophoric points are superposed usually by least-squares fitting.2 – 5 Such methods are generally inadequate for the superposition of structurally dissimilar molecules because the points to be superposed should be predefined, even though the appropriate pairs can automatically be detected.3, 5 In the second approach, various molecular properties are utilized for superposition. These include electron density,6 – 9 molecular volume or shape,10 – 15 charge distribution or molecular electrostatic potential (MEP),16 – 18 hydrophobicity,19, 20 hydrogen bonding capability,2, 3, 21, 22 and so on. The apparently dissimilar molecules often exhibit similar biochemical and pharmacological properties, and thus the propertybased methods may be more suitable for the study of structure-activity relationships. The quality of the superposition can objectively be assessed with the metric of molecular similarity that quantifies the degree of overlap of molecular properties. Concepts and applications of molecular similarity are extensively reviewed in many articles.1, 23 – 25 It has recently been shown that the optimized similarity indices can be correlated directly with activity data.26 – 29 The representative metric for molecular similarity showing its physical concept most rigorously is that of Carbo et al.30 or Hodgkin et al.31 The Carbo similarity index (CAB) of two molecules A and B with respect to a given molecular property P is assessed by

A non-peptide angiotensin II receptor antagonist: 2-butyl-6-methyl-5-(1-oxopyrid-2-yl)-1-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-imidazo[5,4-b]pyridine methanol solvate

The title compound, C 30 H 28 N 8 O.CH 4 O, is one of a series of imidazo[5,4-b]pyridine-based angiotensin II receptor antagonists showing high antihypertensive activity. The overall conformation of the biphenyl-tetrazole moiety as well as its relative orientation with respect to the central fused ring is significantly different from those of related compounds, indicating that this class of compounds has considerable conformational flexibility.