[Scaffold hopping computational approach for searching novel ß-lactamase inhibitors]. / Virtual'nyi skrining s ispol'zovaniem izmeneniia strukturnogo ostova ligandov dlia poiska novykh ingibitorov ß-laktamaz.

We present a novel computational ligand-based virtual screening approach with scaffold hopping capabilities for the identification of novel inhibitors of ß-lactamases which confer bacterial resistance to ß-lactam antibiotics. The structures of known ß-lactamase inhibitors were used as query ligands, and a virtual in silico screening a database of 8 million drug-like compounds was performed in order to select the ligands with similar shape and charge distribution. A set of numerical descriptors was used such as chirality, eigen spectrum of matrices of interatomic distances and connectivity together with higher order moment invariants that showed their efficiency in the field of pattern recognition but have not yet been employed in drug discovery. The developed scaffold-hopping approach was applied for the discovery of analogues of four allosteric inhibitors of serine ß-lactamases. After a virtual in silico screening, the effect of two selected ligands on the activity of TEM type ß-lactamase was studied experimentally. New non-ß-lactam inhibitors were found that showed more effective inhibition of ß-lactamases compared to query ligands.

Novel non-ß-lactam inhibitor of ß-lactamase TEM-171 based on acylated phenoxyaniline.

The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-ß-lactam ß-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial ß-lactamase TEM-171, with a Ki of 88 µM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A ß-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A ß-lactamases. We also hypothesise that the presented route for finding non-ß-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.

Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging.

The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample's electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample--a budding yeast cell (Saccharomyces cerevisiae)--using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution.

Automated identification of crystallographic ligands using sparse-density representations.

A novel procedure for the automatic identification of ligands in macromolecular crystallographic electron-density maps is introduced. It is based on the sparse parameterization of density clusters and the matching of the pseudo-atomic grids thus created to conformationally variant ligands using mathematical descriptors of molecular shape, size and topology. In large-scale tests on experimental data derived from the Protein Data Bank, the procedure could quickly identify the deposited ligand within the top-ranked compounds from a database of candidates. This indicates the suitability of the method for the identification of binding entities in fragment-based drug screening and in model completion in macromolecular structure determination.

Purification, biochemical characterization, and structure of recombinant endo-1,4-ß-xylanase XylE.

The gene xylE encoding endo-1,4-ß-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding ß-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-ß-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

From atoms to proteins.

The investigation of biological macromolecules and the characteristics that determine their function has been of particular interest over the last decades. Here we overview some modern approaches for making the most of the 3-D protein structural information, with a distinctive emphasis on macromolecular crystallography and complementary techniques used to establish the structure-function relationship. A tight link between the biology of the cellular processes and the underlying chemistry of protein function governs the flow of the presented material. The reader will be lead through the basic principles of protein structure analysis and the means to capture the characteristics that portray the function. The techniques exploiting high-resolution data and allowing quantification of molecular motion and structure-activity relationship are given particular attention.

Crystallization and preliminary X-ray analysis of cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens.

A novel cytochrome c nitrite reductase (TvNiR) was isolated from the haloalkalophilic bacterium Thioalkalivibrio nitratireducens. The enzyme catalyses nitrite and hydroxylamine reduction, with ammonia as the only product of both reactions. It consists of 525 amino-acid residues and contains eight haems c. TvNiR crystals were grown by the hanging-drop vapour-diffusion technique. The crystals display cubic symmetry and belong to space group P2(1)3, with unit-cell parameter a = 194 A. A native data set was obtained to 1.5 A resolution. The structure was solved by the SAD technique using the data collected at the Fe absorption peak wavelength.

Modelling bound ligands in protein crystal structures.

Methods for automated identification and building of protein-bound ligands in electron-density maps are described. An error model of the geometrical features of the molecular structure of a ligand based on a lattice distribution of positional parameters is obtained via simulation and is used for the construction of an approximate likelihood scoring function. This scoring function combined with a graph-based search technique provides a flexible model-building scheme and its application shows promising initial results. Several ligands with sizes ranging from 9 to 44 non-H atoms have been identified in various X-ray structures and built in an automatic way using a minimal amount of prior stereochemical knowledge.

The influence of positional errors on the Debye effects.

The relation between a Gaussian perturbation of the atomic positional parameters and the average squared structure-factor amplitude is presented. Using an error-dependent radial distance distribution of an atomic protein model, it can be shown that the Debye effects diminish exponentially as a function of increasing positional errors. These relations can be used to estimate the quality of an atomic model and the corresponding phases. The limiting case of equal atoms with an infinitely large coordinate error results in the classical Wilson model.

Distance distributions and electron-density characteristics of protein models.

The analytical expression for the distribution of an interatomic distance resulting from a known error-free distance and a Gaussian perturbation of the atomic coordinates is presented. This is used to estimate the coordinate error on the basis of known geometric features of protein models via the nearest-neighbour or the radial distance distribution. A simple relation is presented that describes the dependence of the map correlation on the positional error of the protein model, the resolution of the X-ray data and the overall atomic displacement parameter. The distribution of geometrical features and the relation between the map correlation and the positional error can be used in assisting the decision-making process during automated model-building procedures.

Atomic resolution structures of ribonuclease A at six pH values.

The diffraction pattern of protein crystals extending to atomic resolution guarantees a very accurate picture of the molecular structure and enables the study of subtle phenomena related to protein functionality. Six structures of bovine pancreatic ribonuclease at the pH* values 5.2, 5.9, 6.3, 7.1, 8.0 and 8.8 and at resolution limits in the range 1.05-1.15A have been refined. An overall description of the six structures and several aspects, mainly regarding pH-triggered conformational changes, are described here. Since subtle variations were expected, a thorough validation assessment of the six refined models was first carried out. Some stereochemical parameters, such as the N[bond]C(alpha)[bond]C angle and the pyramidalization at the carbonyl C atoms, indicate that the standard target values and their weights typically used in refinement may need revision. A detailed comparison of the six structures has provided experimental evidence on the role of Lys41 in catalysis. Furthermore, insights are given into the structural effects related to the pH-dependent binding of a sulfate anion, which mimics the phosphate group of RNA, in the active site. Finally, the results support a number of thermodynamic and kinetic experimental data concerning the role of the disulfide bridge between Cys65 and Cys72 in the folding of RNase A.

Crystal structure of manganese catalase from Lactobacillus plantarum.

BACKGROUND: Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex. RESULTS: The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase. CONCLUSIONS: A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.

ARP/wARP and molecular replacement.

The aim of ARP/wARP is improved automation of model building and refinement in macromolecular crystallography. Once a molecular-replacement solution has been obtained, it is often tedious to refine and rebuild the initial (search) model. ARP/wARP offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an improved map and (iii) docking of a structure onto a new (or mutated) sequence, followed by rebuilding and refining the side chains in real space. A few examples are presented where ARP/wARP made a considerable difference in the speed of structure solution and/or made possible refinement of otherwise difficult or uninterpretable maps. The resolution range allowing complete autobuilding of protein structures is currently 2.0 A, but for map improvement considerable advances over more conventional refinement techniques are evident even at 3.2 A spacing.

On the enzymatic activation of NADH.

Atomic (1 A) resolution x-ray structures of horse liver alcohol dehydrogenase in complex with NADH revealed the formation of an adduct in the active site between a metal-bound water and NADH. Furthermore, a pronounced distortion of the pyridine ring of NADH was observed. A series of quantum chemical calculations on the water-nicotinamide adduct showed that the puckering of the pyridine ring in the crystal structures can only be reproduced when the water is considered a hydroxide ion. These observations provide fundamental insight into the enzymatic activation of NADH for hydride transfer.

The structures of Escherichia coli inorganic pyrophosphatase complexed with Ca(2+) or CaPP(i) at atomic resolution and their mechanistic implications.

Two structures of Escherichia coli soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate (CaPP(i)-EPPase) and with Ca(2+) (Ca(2+)-EPPase) have been solved at 1.2 and 1.1 A resolution, respectively. In the presence of Mg(2+), this enzyme cleaves pyrophosphate (PP(i)) into two molecules of orthophosphate (P(i)). This work has enabled us to locate PP(i) in the active site of the inorganic pyrophosphatases family in the presence of Ca(2+), which is an inhibitor of EPPase.Upon PP(i) binding, two Ca(2+) at M1 and M2 subsites move closer together and one of the liganded water molecules becomes bridging. The mutual location of PP(i) and the bridging water molecule in the presence of inhibitor cation is catalytically incompetent. To make a favourable PP(i) attack by this water molecule, modelling of a possible hydrolysable conformation of PP(i) in the CaPP(i)-EPPase active site has been performed. The reasons for Ca(2+) being the strong PPase inhibitor and the role in catalysis of each of four metal ions are the mechanistic aspects discussed on the basis of the structures described.

Current state of automated crystallographic data analysis.

A goal of structural biology--and of structural genomics in particular--is to improve the underlying methodology for high-throughput determination of three-dimensional structures of biological macromolecules. Here we address issues related to the development, automation and streamlining of the process of macromolecular X-ray crystal structure solution.

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase.

A significant improvement in the X-ray resolution of crystals of Escherichia coli inorganic pyrophosphatase at cryotemperature was obtained as a result of studying the relationship between the crystal order and cryosolution component concentrations. To perform the experiments, the ability to reverse the flash-cooling process and to return a crystal to ambient temperature was used. In each cycle, the crystal was transferred from a cold nitrogen-gas stream to a cryosolution with modified concentrations of the components. The crystal was then flash-cooled again and the diffraction quality checked. Such a technique allows the screening of a wide concentration range rather quickly without using a large number of crystals and allows the determination of optimal cryosolution component concentrations. The resolution limit for crystals of pyrophosphatase increased by almost 0.7 A, from 1.8 to 1.15 A.

Roles of his205, his296, his303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase.

The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.

Protein titration in the crystal state.

Proteins are complex structures whose overall stability critically depends on a delicate balance of numerous interactions of similar strength, which are markedly influenced by their environment. Here, we present an analysis of the effect of pH on a protein structure in the crystalline state using RNase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induced in the protein. Atomic resolution X-ray diffraction data were collected for crystals at six pH* values ranging from 5.2 to 8.8, and the six independently refined structures reveal subtle, albeit well-defined variations directly related to the pH titration of the protein. The deprotonation of the catalytic His12 residue is clearly evident in the electron density maps, confirming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote from the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistry of the six structures provided accurate estimates of p Kavalues of most of the histidine residues. This study gives further evidence for the advantage of atomic resolution X-ray crystallographic analyses for revealing small but significant structural changes which provide clues to the function of a biological macromolecule.