Lipid exchange in crystal-confined fatty acid binding proteins: X-ray evidence and molecular dynamics explanation.

Fatty acid binding proteins (FABPs) are responsible for the long-chain fatty acids (FAs) transport inside the cell. However, despite the years, since their structure is known and the many studies published, there is no definitive answer about the stages of the lipid entry-exit mechanism. Their structure forms a ß -barrel of 10 anti-parallel strands with a cap in a helix-turn-helix motif, and there is some consensus on the role of the so-called portal region, involving the second α -helix from the cap ( α 2), ß C- ß D, and ß E- ß F turns in FAs exchange. To test the idea of a lid that opens, we performed a soaking experiment on an h-FABP crystal in which the cap is part of the packing contacts, and its movement is strongly restricted. Even in these conditions, we observed the replacement of palmitic acid by 2-Bromohexadecanoic acid (Br-palmitic acid). Our MD simulations reveal a two-step lipid entry process: (i) The travel of the lipid head through the cavity in the order of tens of nanoseconds, and (ii) The accommodation of its hydrophobic tail in hundreds to thousands of nanoseconds. We observed this even in the cases in which the FAs enter the cavity by their tail. During this process, the FAs do not follow a single trajectory, but multiple ones through which they get into the protein cavity. Thanks to the complementary views between experiment and simulation, we can give an approach to a mechanistic view of the exchange process.

Molecular dynamics simulation of the heart type fatty acid binding protein in a crystal environment.

Crystallographic data comes from a space-time average over all the unit cells within the crystal, so dynamic phenomena do not contribute significantly to the diffraction data. Many efforts have been made to reconstitute the movement of the macromolecules and explore the microstates that the confined proteins can adopt in the crystalline network. We explored different strategies to simulate a heart fatty acid binding protein (H-FABP) crystal by means of Molecular Dynamics (MD) simulations. We evaluate the effect of introducing restraints according to experimental isotropic B-factors and we analyzed the H-FABP motions in the crystal using Principal Component Analysis (PCA), isotropic and anisotropic B-factors. We compared the behavior of the protein simulated in the crystal confinement versus in solution, and we observed the effect of that confinement in the mobility of the protein residues. Restraining one-third of Cα atoms based on experimental B-factors produce lower B-factors than simulations without restraints, showing that the position restraint of the atoms with the lowest experimental B-factor is a good strategy to maintain the geometry of the crystal with an obvious decrease in the degrees of motion of the protein. PCA shows that, as position restraint reduces the conformational space explored by the system, the motion of the crystal is better recovered, for an essential subspace of the same size, in the simulations without restraints. Restraining only one Cα seems to be a good balance between giving flexibility to the system and preserving its structure. Communicated by Ramaswamy H. Sarma.

Discovery of N-Substituted 3-Amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic Acids as Highly Potent Third-Generation Inhibitors of Human Arginase I and II.

Recent efforts to identify new highly potent arginase inhibitors have resulted in the discovery of a novel family of (3R,4S)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid analogues with up to a 1000-fold increase in potency relative to the current standards, 2-amino-6-boronohexanoic acid (ABH) and N-hydroxy-nor-l-arginine (nor-NOHA). The lead candidate, with an N-2-amino-3-phenylpropyl substituent (NED-3238), example 43, inhibits arginase I and II with IC50 values of 1.3 and 8.1 nM, respectively. Herein, we report the design, synthesis, and structure-activity relationships for this novel series of inhibitors, along with X-ray crystallographic data for selected examples bound to human arginase II.

Publisher Correction: Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.

Structural Basis of Outstanding Multivalent Effects in Jack Bean α-Mannosidase Inhibition.

Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic-level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high-resolution crystal structures of the Jack bean α-mannosidase (JBα-man) in apo and inhibited states. The three-dimensional structure of JBα-man in complex with the multimeric cyclopeptoid-based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants.

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

New insights into the enzymatic mechanism of human chitotriosidase (CHIT1) catalytic domain by atomic resolution X-ray diffraction and hybrid QM/MM.

Chitotriosidase (CHIT1) is a human chitinase belonging to the highly conserved glycosyl hydrolase family 18 (GH18). GH18 enzymes hydrolyze chitin, an N-acetylglucosamine polymer synthesized by lower organisms for structural purposes. Recently, CHIT1 has attracted attention owing to its upregulation in immune-system disorders and as a marker of Gaucher disease. The 39 kDa catalytic domain shows a conserved cluster of three acidic residues, Glu140, Asp138 and Asp136, involved in the hydrolysis reaction. Under an excess concentration of substrate, CHIT1 and other homologues perform an additional activity, transglycosylation. To understand the catalytic mechanism of GH18 chitinases and the dual enzymatic activity, the structure and mechanism of CHIT1 were analyzed in detail. The resolution of the crystals of the catalytic domain was improved from 1.65 Š(PDB entry 1waw) to 0.95-1.10 Šfor the apo and pseudo-apo forms and the complex with chitobiose, allowing the determination of the protonation states within the active site. This information was extended by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. The results suggest a new mechanism involving changes in the conformation and protonation state of the catalytic triad, as well as a new role for Tyr27, providing new insights into the hydrolysis and transglycosylation activities.

Discovery of (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid and congeners as highly potent inhibitors of human arginases I and II for treatment of myocardial reperfusion injury.

Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structure-activity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Neutron structure of type-III antifreeze protein allows the reconstruction of AFP-ice interface.

Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.

Neutron macromolecular crystallography with LADI-III.

At the Institut Laue-Langevin, a new neutron Laue diffractometer LADI-III has been fully operational since March 2007. LADI-III is dedicated to neutron macromolecular crystallography at medium to high resolution (2.5-1.5 Å) and is used to study key H atoms and water structure in macromolecular structures. An improved detector design and readout system has been incorporated so that a miniaturized reading head located inside the drum scans the image plate. From comparisons of neutron detection efficiency (DQE) with the original LADI-I instrument, the internal transfer of the image plates and readout system provides an approximately threefold gain in neutron detection. The improved performance of LADI-III, coupled with the use of perdeuterated biological samples, now allows the study of biological systems with crystal volumes of 0.1-0.2 mm(3), as illustrated here by the recent studies of type III antifreeze protein (AFP; 7 kDa). As the major bottleneck for neutron macromolecular studies has been the large crystal volumes required, these recent developments have led to an expansion of the field, extending the size and the complexity of the systems that can be studied and reducing the data-collection times required.

Structure and interactions of fish type III antifreeze protein in solution.

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.

Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies.

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D(2)O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 A from a crystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of 0.23 mm(3).

Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein.

The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D(2)O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 A were obtained using a ;radically small' perdeuterated AFP D crystal of dimensions 0.70 x 0.55 x 0.35 mm, corresponding to a volume of 0.13 mm(3).

Electro-optical properties characterization of fish type III antifreeze protein.

Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa-or 14-kDa tandem-globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated.

Design and synthesis of highly potent and selective (2-arylcarbamoyl-phenoxy)-acetic acid inhibitors of aldose reductase for treatment of chronic diabetic complications.

Recent efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel series of highly potent and selective (2-arylcarbamoyl-phenoxy)-acetic acid aldose reductase inhibitors. The compound class features a core template that utilizes an intramolecular hydrogen bond to position the key structural elements of the pharmacophore in a conformation, which promotes a high binding affinity. The lead candidate, example 40, 5-fluoro-2-(4-bromo-2-fluoro-benzylthiocarbamoyl)-phenoxyacetic acid, inhibits aldose reductase with an IC(50) of 30 nM, while being 1100 times less active against aldehyde reductase, a related enzyme involved in the detoxification of reactive aldehydes. In addition, example 40 lowers nerve sorbitol levels with an ED(50) of 31 mg/kg/d po in the 4-day STZ-induced diabetic rat model.

Electrostatic complementarity in an aldose reductase complex from ultra-high-resolution crystallography and first-principles calculations.

The electron density and electrostatic potential in an aldose reductase holoenzyme complex have been studied by density functional theory (DFT) and diffraction methods. Aldose reductase is involved in the reduction of glucose in the polyol pathway by using NADPH as a cofactor. The ultra-high resolution of the diffraction data and the low thermal-displacement parameters of the structure allow accurate atomic positions and an experimental charge density analysis. Based on the x-ray structural data, order-N DFT calculations have been performed on subsets of up to 711 atoms in the active site of the molecule. The charge density refinement of the protein was performed with the program MOPRO by using the transferability principle and our database of charge density parameters built from crystallographic analyses of peptides and amino acids. Electrostatic potentials calculated from the charge density database, the preliminary experimental electron density analysis, DFT computations, and atomic charges taken from the amber software dictionary are compared. The electrostatic complementarity between the cofactor NADP+ and the active site shows up clearly. The anchoring of the inhibitor is due mainly to hydrophobic forces and to only two polar interaction sites within the enzyme cavity. The potentials calculated by x-ray and DFT techniques agree reasonably well. At the present stage of the refinement, the potentials obtained directly from the database are in excellent agreement with the experimental ones. In addition, these results demonstrate the significant contribution of electron lone pairs and of atomic polarization effects to the host and guest mechanism.