Alberto Podjarny

Crystal structure of a eukaryotic initiator tRNA

OUR understanding of the molecular structure–function relationship in tRNA rests mainly on three types of information. First, on the common sequence patterns which have emerged from careful examination of many primary structures1–3; second, a wide variety of spectral and other physical and chemical results must be accounted for by the molecular structure4–6; and third, there is the detailed image of the yeast tRNAPhe molecule independently determined and refined from two different—albeit similar—crystal forms7–10. It is also clear, however, that the molecular model deduced from the yeast tRNAphe crystal structure cannot be easily reconciled with all structural requirements for function and is best considered a well-defined and stable canonical form of tRNA which is packed in an unusually well-ordered way in specific crystal lattices. Notwithstanding the enormous value of this canonical form in explaining the basic architectural features of tRNA, it is clearly important to image other crystalline tRNAs; particularly tRNAs that exhibit different functions (such as, initiators) or have significantly different covalent structures (for example, class III tRNAs)1 or those that crystallise in different solvent conditions. We report here the initial results of the crystal structure determination of a eukaryotic initiator tRNA crystallised from a highly polar aqueous solvent11,12. Its architecture is essentially the same as crystalline yeast tRNAphe, except for a small but significant difference in the position of the anticodon arm.

Neutron crystallography: opportunities, challenges, and limitations.

Neutron crystallography has had an important, but relatively small role in structural biology over the years. In this review of recently determined neutron structures, a theme emerges of a field currently expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times, and employing more sophisticated structure determination and refinement methods. The origin of this transformation can be found in a number of advances including first, the development of neutron image-plates and quasi-Laue methods at nuclear reactor neutron sources and the development of time-of-flight Laue methods and electronic detectors at spallation neutron sources; second, new facilities and methods for sample perdeuteration and crystallization; third, new approaches and computational tools for structure determination.

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.

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X‐ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK‐860) and Minalrestat (WAY‐509) were determined at atomic resolutions of 0.92 Å and 1.1 Å, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion‐binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestats hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo‐fluorobenzyl group inside the “specificity” pocket. The interactions between Minalrestats bromo‐fluorobenzyl group and the enzyme include the stacking against the side‐chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main‐chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active‐site residue His110. The 1′‐position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nϵ2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H‐atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl‐ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design. Proteins 2004.

Modulation of aldose reductase inhibition by halogen bond tuning.

In this paper, we studied a designed series of aldose reductase (AR) inhibitors. The series was derived from a known AR binder, which had previously been shown to form a halogen bond between its bromine atom and the oxygen atom of the Thr-113 side chain of AR. In the series, the strength of the halogen bond was modulated by two factors, namely bromine-iodine substitution and the fluorination of the aromatic ring in several positions. The role of the single halogen bond in AR-ligand binding was elucidated by advanced binding free energy calculations involving the semiempirical quantum chemical Hamiltonian. The results were complemented with ultrahigh-resolution X-ray crystallography and IC50 measurements. All of the AR inhibitors studied were shown by X-ray crystallography to bind in an identical manner. Further, it was demonstrated that it was possible to decrease the IC50 value by about 1 order of magnitude by tuning the strength of the halogen bond by a monoatomic substitution. The calculations revealed that the protein-ligand interaction energy increased upon the substitution of iodine for bromine or upon the addition of electron-withdrawing fluorine atoms to the ring. However, the effect on the binding affinity was found to be more complex due to the change of the solvation/desolvation properties within the ligand series. The study shows that it is possible to modulate the strength of a halogen bond in a protein-ligand complex as was designed based on the previous studies of low-molecular-weight complexes.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Å, 100K; 0.80 Å, 15K; 1.75 Å, 293K), neutron Laue data (2.2 Å, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

The p53 pro-apoptotic tumour suppressor is mutated or functionally altered in most cancers. In epithelial tumours induced by ‘high-risk’ mucosal human papilloma viruses, including human cervical carcinoma and a growing number of head-and-neck cancers, p53 is degraded by the viral oncoprotein E6 (ref. 2). In this process, E6 binds to a short leucine (L)-rich LxxLL consensus sequence within the cellular ubiquitin ligase E6AP. Subsequently, the E6/E6AP heterodimer recruits and degrades p53 (ref. 4). Neither E6 nor E6AP are separately able to recruit p53 (refs 3, 5), and the precise mode of assembly of E6, E6AP and p53 is unknown. Here we solve the crystal structure of a ternary complex comprising full-length human papilloma virus type 16 (HPV-16) E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6–p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumour suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against oncogenesis mediated by human papilloma virus.

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

Abstract.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.

Virtual screening for inhibitors of human aldose reductase

The inhibition of aldose reductase (AR) provides an interesting strategy to prevent the complications of chronic diabetes. Although a large number of different AR inhibitors are known, very few of these compounds exhibit sufficient efficacy in clinical trials. We performed a virtual screening based on the ultrahigh resolution crystal structure of the inhibitor IDD594 in complex with human AR. AR operates on a large scale of structurally different substrates. To achieve this pronounced promiscuity, the enzyme can adapt rather flexibly to its substrates. Likewise, it has a similar adaptability for the binding of inhibitors. We applied a protocol of consecutive hierarchical filters to search the Available Chemicals Directory. In the first selection step, putative ligands were chosen that exhibit functional groups to anchor the anion‐binding pocket of AR. Subsequently, a pharmacophore model based on the binding geometry of IDD594 and the mapping of the binding pocket in terms of putative “hot spots” of binding was applied as a second consecutive filter. In a third and final filtering step, the remaining candidate molecules were flexibly docked into the binding pocket of IDD594 with FlexX and ranked according to their estimated DrugScore values. Out of 206 compounds selected by this search and complemented by a cluster analysis and visual inspection, 9 compounds were selected and subjected to biological testing. Of these, 6 compounds showed IC50 values in the micromolar range. According to the proposed binding mode, the two inhibitors BTB02809 (IC50 = 2.4 ± 0.5 μM) and JFD00882 (IC50 = 4.1 ± 1.0 μM) both place a nitro group into the hydrophobic specificity pocket of human AR in an orientation coinciding with the position of the bromine atom of IDD594. The interaction of this Br with Thr113 has been identified as a key feature that is responsible for selectivity enhancement. Proteins 2004.

Charge Density and Electrostatic Interactions of Fidarestat, an Inhibitor of Human Aldose Reductase

The charge density and the topological features of fidarestat, an inhibitor of human aldose reductase, have been determined from ultra high-resolution X-ray diffraction data at 100 K. The modeled electron density was used to calculate the electrostatic interaction energy of fidarestat and its (2R,4S) stereoisomer with the human aldose reductase by using the ELMAM database as coded in the MoPro program. Such calculation may be extended to other protein complexes for which accurate high resolution X-ray data are available. The paper also discusses the hydrogen bonds in the fidarestat crystal. There are notably two hydrogen bonds with a pi system as an acceptor. All the chemical bonds and the intermolecular interactions, especially these two pi...H bonds, have been quantitatively studied by topological analysis. The three-dimensional electrostatic potential calculated on the molecular surface emphasizes the preferential polar binding sites of fidarestat. Theses interacting features in the molecule are crucial for drug-receptor recognition. The interactions between chemical groups in the crystal are also analyzed by computing the electrostatic energy using the latest advancements of the MoPro crystallographic software. The complexes of fidarestat and its (2R,4S) stereoisomer with human aldose reductase were modeled with a multipolar atom model transferred from our experimental electron density database. Accurate estimation of electrostatic interaction energy between inhibitors and the main residues of the protein active site is derived from this high detail level of the electron density.