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.

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.

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 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.

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.

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.

Subatomic and atomic crystallographic studies of aldose reductase: implications for inhibitor binding.

The determination of several of aldose reductase-inhibitor complexes at subatomic resolution has revealed new structural details, including the specific interatomic contacts involved in inhibitor binding. In this article, we review the structures of the complexes of ALR2 with IDD 594 (resolution: 0.66 angstrom, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P2(1)), IDD 393 (resolution: 0.90 angstrom, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 angstrom, IC50: 9 nM, space group: P2(1)) and minalrestat (resolution: 1.10 angstrom, IC50: 73 nM, space group: P1). The structures are compared and found to be highly reproductible within the same space group (root mean square (RMS) deviations: 0.15 approximately 0.3 angstrom). The mode of binding of the carboxylate inhibitors IDD 594 and IDD 393 is analysed. The binding of the carboxylate head can be accurately determined by the subatomic resolution structures, since both the protonation states and the positions of the atoms are very precisely known. The differences appear in the binding in the specificity pocket. The high-resolution structures explain the differences in IC50, which are confirmed both experimentally by mass spectrometry measures of VC50 and theoretically by free energy perturbation calculations. The binding of the cyclic imide inhibitors fidarestat and minalrestat is also described, focusing on the observation of a Cl(-) ion which binds simultaneously with fidarestat. The presence of this anion, binding also to the active site residue His110, leads to a mechanism in which the inhibitor can bind in a neutral state and then become charged inside the active site pocket. This mechanism can explain the excellent in vivo properties of cyclic imide inhibitors. In summary, the complete and detailed information supplied by the subatomic resolution structures can explain the differences in binding energy of the different inhibitors.

Low-resolution data analysis for low-density lipoprotein particle.

The knowledge of the molecular structure of LDL, a large lipoprotein complex, is of great interest for medical investigations. Currently available LDL crystals do not diffract to high resolution and do not allow the application of standard crystallographic techniques. Additional difficulties arise because of a very dense crystal packing and the presence of several components with quite different mean densities. Several ab initio phasing methods previously reported by the authors have been successfully applied to find a crystallographic image of LDL at a resolution of 27 A. The most promising results have been obtained using direct phasing with a connectivity analysis of the electron-density maps. The current image makes it possible to discern a single particle covered by a layer of relatively high density that is asymmetrically distributed on the particle surface. It shows a partition of high and low densities inside the particle and, in particular, strips of varying density in the lipid core.

Aldose and aldehyde reductases: correlation of molecular modeling and mass spectrometric studies on the binding of inhibitors to the active site.

Aldose and aldehyde reductases are monomeric NADPH-dependent oxidoreductases that catalyze the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The overall three-dimensional structures of the enzymes are composed of similar alpha/beta TIM-barrels, and the active site residues Tyr 50, His 113, and Trp 114 interacting with the hydrophilic heads of inhibitors are conserved. We have used molecular modeling and mass spectrometry to characterize the interactions between the enzymes and three aldose reductase inhibitors: tolrestat, sorbinil, and zopolrestat. Unlike the IC(50) values (concentration of inhibitor giving 50% of inhibition in solution), the Vc(50) values measured by mass spectrometry (accelerating voltage of ions needed to dissociate 50% of a noncovalent complex in the gas phase) for the two enzymes are similar, and they correlate with the electrostatic and hydrogen-bonding energies calculated between the conserved Tyr 50, His 113, and Trp 114 and the inhibitors. The results of our comparison agree with detailed structural information obtained by X-ray crystallography, suggesting that nonconserved residues from the C-terminal loop account for differences in IC(50) values for the two enzymes. Additionally, they confirm our previous assumption that the Vc(50) values reflect the enzyme-inhibitor electrostatic and hydrogen-bonding interactions and exclude the hydrophobic interactions.

Low-resolution ab initio phasing: problems and advances.

If only native amplitudes are used for structure determination, then additional 'theoretical' information is necessary to determine their phases. For use in a phasing procedure, this information can be formulated as a selection criterion (figure of merit) which assigns a reliability weight to every trial phase set and distinguishes the closest ones to the true phase set. Different types of additional information may be tested as a selection criterion: electron-density histograms, connectivity properties, statistical likelihood, atomicity etc. A common feature of such criteria is that they do not unambiguously judge the phase quality at low resolution. Nevertheless, the selection of the phase sets with best criterion values increases the ratio of good phase sets in the ensemble considered. An approximate solution of the phase problem may then be found by averaging the selected phase sets. Cluster analysis of the selected phase sets and averaging within clusters allow further improvement of this solution.

Density constraints and low-resolution phasing.

Direct phasing needs additional information of a non-specific kind in order to select the correct phase set from all possible ones. This paper analyses the use of constraints which can be formulated in terms of electron-density values. One- and multi-dimensional histograms and connectivity properties are implemented as such constraints in density-modification procedures. These approaches usually cannot unambiguously select the best solution from a set of alternative phase variants. Nevertheless, they do allow the rejection of wrong solutions and the use of cluster analysis and averaging on the remaining variants provide a good starting point for further phase-refinement procedures.

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.

The structure of human aldose reductase bound to the inhibitor IDD384.

The crystallographic structure of the complex between human aldose reductase (AR2) and one of its inhibitors, IDD384, has been solved at 1.7 A resolution from crystals obtained at pH 5.0. This structure shows that the binding of the inhibitor's hydrophilic head to the catalytic residues Tyr48 and His110 differs from that found previously with porcine AR2. The difference is attributed to a change in the protonation state of the inhibitor (pK(a) = 4.52) when soaked with crystals of human (at pH 5.0) or pig lens AR2 (at pH 6.2). This work demonstrates how strongly the detailed binding of the inhibitor's polar head depends on its protonation state.

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.

Production of crystals of human aldose reductase with very high resolution diffraction.

As the action of human aldose reductase (hAR) is thought to be linked to the pathogenesis of diabetic complications, much effort has been directed towards the analysis of the catalytic mechanism and the development of specific inhibitors. Here, the crystallization of recombinant hAR with its cofactor NADP+ at 277 K in the presence of the precipitating agent PEG 6000 is reported. The crystals diffract to high resolution (1.1 A) and belong to the P21 space group with unit-cell parameters a = 49.97, b = 67.14, c = 48. 02 A, beta = 92.2 degrees with one molecule per asymmetric unit. Seleno-substituted hAR crystals were also produced and diffract to 1. 7 A on a conventional X-ray source.

On the ab initio solution of the phase problem or macromolecules at very low resolution. II. Generalized likelihood based approach to cluster discrimination.

The multisolution strategies for direct phasing at very low resolution, such as the few atoms model technique, result in a number of alternative phase sets, each of them arising from a cluster of closely related models. Use of a Monte-Carlo type computer procedure is suggested to choose between the possible phase sets. It consists of generating a large number of pseudo-atom models inside the mask defined by a trial phase set and the use of histograms of magnitude correlation to evaluate the masks. It is shown that the procedure may be considered as a generalization of the statistical maximum-likelihood principle and may be used as a powerful supplementary tool in the likelihood-based approaches to the phase problem solution.

A multicopy modeling of the water distribution in macromolecular crystals.

A multicopy protocol is proposed for modeling macromolecular hydration using diffraction experimental data (X-ray or neutron) to search for a better description of delocalized water sites than that given by point water models. The model consists of one macro-molecule and several copies of each water molecule, refined simultaneously against diffraction data using molecular dynamics techniques. The protocol was applied to BPTI and an RNA tetradecamer. The sites defined by the different copies range from very ordered ones to continuous channels; they fit the density maps and agree with the diffraction amplitudes with an accuracy comparable with usual crystallographic methods. The delocalization of water in channels agrees with the high mobility observed in NMR experiments.

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.