Perdeuteration: improved visualization of solvent structure in neutron macromolecular crystallography.

The 1.8 Šresolution neutron structure of deuterated type III antifreeze protein in which the methyl groups of leucine and valine residues are selectively protonated is presented. Comparison between this and the 1.85 Šresolution neutron structure of perdeuterated type III antifreeze protein indicates that perdeuteration improves the visibility of solvent molecules located in close vicinity to hydrophobic residues, as cancellation effects between H atoms of the methyl groups and nearby heavy-water molecules (D2O) are avoided.

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp).

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

A 'specificity' pocket inferred from the crystal structures of the complexes of aldose reductase with the pharmaceutically important inhibitors tolrestat and sorbinil.

BACKGROUND: Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket. RESULTS: The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase. CONCLUSIONS: The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution.

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Šresolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.

Crystal structure of the nucleosome core particle at 16 A resolution.

The crystal structure of the nucleosome core particle has been studied by neutron diffraction to a resolution of 16 A. By using H2O/D2O solvent contrast variation, the structures of the DNA and histone core were analysed separately. The DNA, as seen at this resolution, forms a super-helix of pitch 25.8 A, radius 42.1 A and 1.8 turns in length. The histone core itself is approximately helical and follows the DNA along the inside of the super-helix, giving the nucleosome core particle an overall 2-fold axis of symmetry. Four regions can be distinguished in the protein density, which we interpret as dimers of histones within the octameric core. The dimers have been assigned on the basis of other evidence as being of two kinds, (H2A-H2B) and (H3-H4). Because solvent contrast variation can distinguish between hydrophobic and hydrophilic regions in the protein density, our results suggest that the interface between the monomers of each dimer is probably quite hydrophobic in character, while the interaction between dimers is weaker and/or more hydrophilic. The protein is in contact with most of the DNA and there are some regions where it may penetrate between the turns of the super-helix. In particular, the tetramer (H4-H3)-(H3-H4) is in close contact with the central part of the DNA, but significant contacts are seen also between the histones H3 and the extremities of the super-helix, thus explaining the stability of a nucleosome-like particle depleted of H2A and H2B. Significant departures from the molecular 2-fold axis of symmetry occur in the relative arrangements of the two (H2A-H2B) dimers.

Crystallographic structure of an RNA helix: [U(UA)6A]2.

The crystallographic structure of the synthetic oligoribonucleotide, U(UA)6A, has been solved at 2.25 A resolution. The crystallographic refinement permitted the identification of 91 solvent molecules, with a final agreement factor of 13%. The molecule is a dimer of 14 base-pairs and shows the typical features of an A-type helix. However, the presence of two kinks causes a divergence from a straight helix. The observed deformation, which is stabilized by a few hydrogen bonds in the crystal packing, could be due to the relatively high (35 degrees C) temperature of crystallization. The complete analysis of the structure is presented. It includes the stacking geometries, the backbone conformation and the solvation.

Ultrahigh resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 A.

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.

Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies.

Aldose reductase is a NADP(H)-dependent enzyme, believed to be strongly implicated in the development of degenerative complications of Diabetes Mellitus. The search for specific inhibitors of this enzyme has thus become a major pharmaceutic challenge. In this study, we applied both X-ray crystallography and mass spectrometry to characterize the interactions between aldose reductase and four representative inhibitors: AminoSNM, Imirestat, LCB3071, and IDD384. If crystallography remains obviously the only way to get an extensive description of the contacts between an inhibitor and the enzymatic site, the duration of the crystallographic analysis makes this technique incompatible with high throughput screenings of inhibitors. On the other hand, dissociation experiments monitored by mass spectrometry permitted us to evaluate rapidly the relative gas-phase stabilities of the aldose reductase-inhibitor noncovalent complexes. In our experiments, dissociation in the gas-phase was provoked by increasing the accelerating voltage of the ions (Vc) in the source-analyzer interface region: the Vc value needed to dissociate 50% of the noncovalent complex initially present (Vc50) was taken as a gas-phase stability parameter of the enzyme-inhibitor complex. Interestingly, the Vc50 were found to correlate with the energy of the electrostatic and H-bond interactions involved in the contact aldose reductase/inhibitor (Eel-H), computed from the crystallographic model. This finding may be specially interesting in a context of drug development. Actually, during a drug design optimization phase, the binding of the drug to the target enzyme is often optimized by modifying its interatomic electrostatic and H-bond contacts; because they usually depend on a single atom change on the drug, and are easier to introduce than the hydrophobic interactions. Therefore, the Vc50 may help to monitor the chemical modifications introduced in new inhibitors. X-ray crystallography is clearly needed to get the details of the contacts and to rationalize the design. Nevertheless, once the cycle of chemical modification is engaged, mass spectrometry can be used to select a priori the drug candidates which are worthy of further crystallographic investigation. We thus propose to use the two techniques in a complementary way, to improve the screening of large collections of inhibitors.

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure.

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.

Heavy-atom refinement against solvent-flattened phases.

A new algorithm for refinement of heavy-atom parameters is defined by an iterative procedure where external phases are provided by density modification. This algorithm is applied to two cases, tRNA(Asp) and the complex between tRNA(Asp) and aspartyl-tRNA synthetase. In the first case, where the structure was solved by multiple isomorphous replacement (MIR) methods, it was found that the new method gives accurate values for the native-derivative scale and four occupancy of heavy-atom sites. Position refinement was more delicate and it needed to be handled in a restricted resolution range. In the second case, where a similar method was used in the early stages of solving the phase problem, it slightly decreased the phase error. It was followed by an improvement of the density-modification masks, which led to better maps at higher resolution.

Selectivity determinants of the aldose and aldehyde reductase inhibitor-binding sites.

Aldose reductase and aldehyde reductase belong to the aldo-keto reductase superfamily of enzymes whose members are responsible for a wide variety of biological functions. Aldose reductase has been identified as the first enzyme involved in the polyol pathway of glucose metabolism which converts glucose into sorbitol. Glucose over-utilization through the polyol pathway has been linked to tissue-based pathologies associated with diabetes complications, which make the development of a potent aldose reductase inhibitor an obvious and attractive strategy to prevent or delay the onset and progression of the complications. Structural studies of aldose reductase and the homologous aldehyde reductase in complex with inhibitor were carried out to explain the difference in the potency of enzyme inhibition. The aim of this review is to provide a comprehensive summary of previous studies to aid the development of aldose reductase inhibitors that may have less toxicity problems than the currently available ones.

Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase-inhibitor complex.

Protonation states determination by neutron (2.2 A at room temperature) and X-ray (0.66 A at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW=36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00x0.67x0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B>5 A2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.

High-resolution crystallography and drug design.

Ultra-high-resolution X-ray crystallography of macromolecules (i.e. resolution better than 0.8 Angstroms) is a rising field that promises to provide new insight into the structure-function relationships of biomacromolecules. The picture emerging from macromolecular structures at this resolution is far more complex than previously understood, requiring for its study improved tools for structure refinement, analysis and annotation. Some of these problems were highlighted during the recent High Resolution Drug Design Meeting (Bischenberg-Strasbourg, France, 13-16 May 2004). We will review here some of the results and discussions that took place during that meeting and elaborate on the trends and challenges ahead in this emerging new field of research.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

On the solution of the molecular-replacement problem at very low resolution: application to large complexes.

The applicability of the molecular-replacement (MR) method, implemented through the AMoRe package [Navaza (1994). Acta Cryst. A50, 157-163], is studied at very low resolution (d > 20 A) and for very large molecular complexes. Due to the nature of the low-resolution data, specific problems appear. In particular, rotation-function peaks are very broad and translation functions based on Patterson overlap show large spurious peaks. To solve these problems, the translation function is replaced by a search using amplitude correlation and a systematic three-dimensional angular search is performed around each rotation-function peak. Furthermore, these functions are applied in different resolution ranges during the same search. The corresponding algorithms are applied to two cases: the tRNA(Asp)-synthetase complex (neutron diffraction data) and a ribosome model crystal (calculated data). This new implementation is shown to solve the problem for a variety of search models, ranging from a detailed atomic model to a rough envelope.

Ab initio low-resolution phasing in crystallography of macromolecules by maximization of likelihood.

Statistical likelihood criteria were tested to select the true (or closest to true) structure-factor phases from an ensemble of phase sets. To define the criterion value for a given trial phase set, the trial 'molecular region' is defined as a region consisting of the points with the highest values in the Fourier synthesis calculated with the observed magnitudes and the trial set of phases. The structure studied is considered as composed of atoms randomly placed inside the trial molecular region. The figure of merit is defined as the likelihood corresponding to this hypothesis, i.e. the probability that the structure-factor magnitudes calculated (from the positions of atoms randomly placed into the trial region) are equal to the observed magnitudes. The concept of generalized likelihood is introduced to make the calculations more straightforward. The tests performed for known structures with the use of experimentally observed magnitudes show that in general it is impossible to unambiguously determine the best phases among a 'population' of trial phase sets. Nevertheless, the random generation of a great number of phase sets and the selection of phase sets with high likelihood values give a collection of variants with a higher concentration of 'good' phase sets than those found in the original population. Averaging the selected phase sets gives a starting solution of the low-resolution phase problem.

Approaches to very low resolution phasing of the ribosome 50S particle from Thermus thermophilus by the few-atoms-models and molecular-replacement methods.

Estimates for the phases of the X-ray diffraction data from the 50S ribosomal particle of Thermus thermophilus has been made to an effective resolution around 80 A using the few-atoms-modes ab initio technique [Lunin, Lunina, Petrova, Vernoslova, Urzhumtsev & Podjarny (1995). Acta Cryst. D51, 896-903]. This technique models the density with a small number of Gaussian spheres to generate a large number of possible phase sets and then uses clustering algorithms to identify the best ones. Independently, an envelope obtained from electron-micrograph image reconstruction [Yonath, Leonard & Wittmann (1987). Science, 236, 813-816] was oriented and positioned using the molecular-replacement technique, specially adapted to the very low resolution case [Urzhumtsev & Podjarny (1995). Acta Cryst. D51, 888-895]. The two methods show similar packing arrangements. The electron density calculated by the few-atoms-models technique without any assumption on the number of molecules in asymmetric unit or on their shape shows recognizable features of the particle.