Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of the GyrA subunit of DNA gyrase from Staphylococcus aureus strain Mu50.

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological form of bacterial DNA. In this study, the C-terminal domain of the GyrA subunit of DNA gyrase from Staphylococcus aureus Mu50 strain was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.80 Šresolution using a synchrotron-radiation source. The crystal belonged to space group P2(1), with unit-cell parameters a = 37.28, b = 80.19, c = 50.22 Å, ß = 110.64°. The asymmetric unit contained one molecule, with a corresponding V(M) of 2.02 Å(3) Da(-1) and a solvent content of 39.2%.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the N-terminal domain of DEAD-box RNA helicase from Staphylococcus aureus strain Mu50.

DEAD-box helicases are enzymes with an ATP-dependent RNA-unwinding function that are involved in a variety of cellular processes including RNA splicing, ribosome biogenesis and RNA degradation. In this study, the N-terminal domain of DEAD-box RNA helicase from Staphylococcus aureus strain Mu50 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.60 Šresolution using a synchrotron-radiation source. The crystal belonged to space group P1, with unit-cell parameters a=70.81, b=80.23, c=86.25 Å, α=69.54, ß=66.54, γ=87.32°. The unit cell contained six molecules, with a corresponding VM of 2.91 Å3 Da(-1) and a solvent content of 56.1%.

Binary image representation of a ligand binding site: its application to efficient sampling of a conformational ensemble.

BACKGROUND: Modelling the ligand binding site of a protein is an important component of understanding protein-ligand interactions and is being actively studied. Even if the side chains are restricted to rotamers, a set of commonly-observed low-energy conformations, the exhaustive combinatorial search of ligand binding site conformers is known as NP-hard. Here we propose a new method, ROTAIMAGE, for modelling the plausible conformers for the ligand binding site given a fixed backbone structure. RESULTS: ROTAIMAGE includes a procedure of selecting ligand binding site residues, exhaustively searching rotameric conformers, clustering them by dissimilarities in pocket shape, and suggesting a representative conformer per cluster. Prior to the clustering, the list of conformers generated by exhaustive search can be reduced by pruning the conformers that have near identical pocket shapes, which is done using simple bit operations. We tested our approach by modelling the active-site inhibitor binding pockets of matrix metalloproteinase-1 and -13. For both cases, analyzing the conformers based on their pocket shapes substantially reduced the 'computational complexity' (10 to 190 fold). The subsequent clustering revealed that the pocket shapes of both proteins could be grouped into approximately 10 distinct clusters. At this level of clustering, the conformational space spanned by the known crystal structures was well covered. Heatmap analysis identified a few bit blocks that combinatorially dictated the clustering pattern. Using this analytical approach, we demonstrated that each of the bit blocks was associated with a specific pocket residue. Identification of residues that influenced the shape of the pocket is an interesting feature unique to the ROTAIMAGE algorithm. CONCLUSIONS: ROTAIMAGE is a novel algorithm that was efficient in exploring the conformational space of the ligand binding site. Its ability to identify 'key' pocket residues also provides further insight into conformational flexibility with specific implications for protein-ligand interactions.

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.

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 alpha2 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 alpha2 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 A movement of alpha2. 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.

Crystallization and preliminary X-ray analysis of a truncated mutant of yeast nuclear thiol peroxidase, a novel atypical 2-Cys peroxiredoxin.

Saccharomyces cerevisiae nTPx is a thioredoxin-dependent thiol peroxidase that is localized in the nucleus. nTPx belongs to the C-type atypical 2-Cys peroxiredoxin family members, which are frequently called BCPs or PrxQs. A double mutant (C107S/C112S) of nTPx overexpressed in Escherichia coli was spontaneously degraded upon freezing and thawing and its truncated form (residues 57-215; MW = 17837 Da) was crystallized with PEG 3350 and mercury(II) acetate as precipitants using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.8 A resolution using X-ray synchrotron radiation. The crystals belong to the trigonal space group P3(2), with unit-cell parameters a = b = 37.54, c = 83.26 A. The asymmetric unit contains one molecule of truncated mutant nTPx, with a corresponding VM of 1.91 A3 Da(-1) and a solvent content of 35.5%.

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.

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.

Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins.

Thioredoxin-dependent thiol peroxidase (Tpx) from Escherichia coli represents a group of antioxidant enzymes that are widely distributed in pathogenic bacterial species and which belong to the peroxiredoxin (Prx) family. Bacterial Tpxs are unique in that the location of the resolving cysteine (CR) is different from those of other Prxs. E. coli Tpx (EcTpx) shows substrate specificity toward alkyl hydroperoxides over H2O2 and is the most potent reductant of alkyl hydroperoxides surpassing AhpC and BCP, the other E. coli Prx members. Here, we present the crystal structure of EcTpx in the oxidized state determined at 2.2-A resolution. The structure revealed that Tpxs are the second type of atypical 2-Cys Prxs with an intramolecular disulfide bond formed between the peroxidatic (CP, Cys61) and resolving (Cys95) cysteine residues. The extraordinarily long N-terminal chain of EcTpx folds into a beta-hairpin making the overall structure very compact. Modeling suggests that, in atypical 2-Cys Prxs, the CR-loop as well as the CP-loop may alternately assume the fully folded or locally unfolded conformation depending on redox states, as does the CP-loop in typical 2-Cys Prxs. EcTpx exists as a dimer stabilized by hydrogen bonds. Its substrate binding site extends to the dimer interface. A modeled structure of the reduced EcTpx in complex with 15-hydroperoxyeicosatetraenoic acid suggests that the size and shape of the binding site are particularly suited for long fatty acid hydroperoxides consistent with its greater reactivity.

Crystallization and preliminary X-ray analysis of Escherichia coli p20, a novel thiol peroxidase.

Escherichia coli p20 is a thioredoxin-dependent thiol peroxidase. This protein represents a novel group of antioxidant enzymes that are widely expressed in various pathogenic bacteria and show distant yet significant sequence homology with peroxiredoxins. E. coli p20, overexpressed in E. coli, was crystallized with PEG 4000 and 2-propanol as precipitants using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.2 A resolution using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 38.97, b = 58.97, c = 127.59 A. The asymmetric unit contains two p20 molecules, with a corresponding V(M) of 2.06 A(3) Da(-1) and a solvent content of 40.4%.