A. Joachimiak

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.

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 Å, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P21), IDD 393 (resolution: 0.90 Å, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 Å, IC50: 9 nM, space group: P21) and minalrestat (resolution: 1.10 Å, 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 ∼ 0.3 Å). 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.

The crystallographic structure of the aldose reductase-IDD552 complex shows direct proton donation from tyrosine 48.

The X-ray crystal structure of human aldose reductase (ALR2) in complex with the inhibitor IDD552 was determined using crystals obtained from two crystallization conditions with different pH values (pH 5 and 8). In both structures the charged carboxylic head of the inhibitor binds to the active site, making hydrogen-bond interactions with His110 and Tyr48 and electrostatic interactions with NADP+. There is an important difference between the two structures: the observation of a double conformation of the carboxylic acid moiety of the inhibitor at pH 8, with one water molecule interacting with the main configuration. This is the first time that a water molecule has been observed deep inside the ALR2 active site. Furthermore, in the configuration with the lower occupancy factor the difference electron-density map shows a clear peak (2.5sigma) for the H atom in the hydrogen bond between the inhibitors carboxylic acid and the Tyr48 side-chain O atom. The position of this peak implies that this H atom is shared between both O atoms, indicating possible direct proton transfer from this residue to the inhibitor. This fact agrees with the model of the catalytic mechanism, in which the proton is donated by the Tyr48 hydroxyl to the substrate. These observations are useful both in drug design and in understanding the ALR2 mechanism.

Autotracing of Escherichia coli acetate CoA-transferase α-subunit structure using 3.4 Å MAD and 1.9 Å native data

The automation of protein structure determination is an essential component for high-throughput structural analysis in protein X-ray crystallography and is a key element in structural genomics. This highly challenging undertaking relies at present on the availability of high-quality native and derivatized protein crystals diffracting to high or moderate resolution, respectively. Obtaining such crystals often requires significant effort. The present study demonstrates that phases obtained at low resolution (>3.0 A) from crystals of SeMet-labeled protein can be successfully used for automated structure determination. The crystal structure of acetate CoA-transferase alpha-subunit was solved using 3.4 A multi-wavelength anomalous dispersion data collected from a crystal containing SeMet-substituted protein and 1.9 A data collected from a native protein crystal.

Mitigation of X-ray damage in macromolecular crystallography by submicrometre line focusing.

Reported here are measurements of the penetration depth and spatial distribution of photoelectron (PE) damage excited by 18.6 keV X-ray photons in a lysozyme crystal with a vertical submicrometre line-focus beam of 0.7 µm full-width half-maximum (FWHM). The experimental results determined that the penetration depth of PEs is 5 ± 0.5 µm with a monotonically decreasing spatial distribution shape, resulting in mitigation of diffraction signal damage. This does not agree with previous theoretical predication that the mitigation of damage requires a peak of damage outside the focus. A new improved calculation provides some qualitative agreement with the experimental results, but significant errors still remain. The mitigation of radiation damage by line focusing was measured experimentally by comparing the damage in the X-ray-irradiated regions of the submicrometre focus with the large-beam case under conditions of equal exposure and equal volumes of the protein crystal, and a mitigation factor of 4.4 ± 0.4 was determined. The mitigation of radiation damage is caused by spatial separation of the dominant PE radiation-damage component from the crystal region of the line-focus beam that contributes the diffraction signal. The diffraction signal is generated by coherent scattering of incident X-rays (which introduces no damage), while the overwhelming proportion of damage is caused by PE emission as X-ray photons are absorbed.

X-ray-induced cooperative atomic movements in protein crystals

C655 The XIPHOS system in Durham is now operational, allowing crystal structures to be determined routinely at temperatures down to 2K. [1] In order to reach these temperatures, the sample is isolated from the environment using vacuum shrouds made from beryllium. These shrouds contain unevenly distributed crystalline parts, resulting in powder rings of non-uniform intensity. When collecting data with a weakly diffracting crystal where the reflections from the beryllium are strong in comparison to those reflections from the crystal, the uneven intensities from the rings can distort the intensities calculated by integration programs. This can be avoided, and improved data quality obtained by masking out the regions of the diffraction image which are affected by beryllium scattering. Any reflections which overlap with the masked region are discarded by the integration program. As the scattering from the beryllium shrouds and the crystal originates from different positions, overlapping reflections can be separated by changing the detector distance. By using masks and recovering the lost data with these different detector distances, data quality can be improved compared with data obtained at one distance and without masking. Masquerade has been written to generate the mask files required for this technique quickly and accurately. The positions of the beryllium rings are calculated using an ab-initio model of the position of the shrouds in 3D space, and include goniometer rotations as well as centring offsets for the shrouds themselves. The program is written in C++ and uses multithreading to make full use of modern multi-core processors. It runs on a variety of platforms and can generate thousands of mask images for a full data collection in minutes. The use of this program along with the data collection at multiple detector distances has been found to improve the data quality obtained from weakly diffracting crystals, showing clear improvements in R1, wR2 and Rsigma values over data integrated without the masks.

Inhibitor binding to aldose reductase studied at subatomic resolution

Resolution Alberto Podjarny, Andre Mitschler, Isabelle Hazemann, Tania Petrova, Federico Ruiz, Eduardo Howard, Connie Darmanin, Roland Chung, Thomas R. Schneider, Ruslan Sanishvili, Clemens Schulze-Briesse, Takashi Tomizaki, Michael Van Zandt, Mitsuru Oka, Andrzej Joachimiak, Ossama El-Kabbani, IGBMC, CNRS, Illkirch, France. Department of Medicinal Chemistry, Monash U., Australia. FIRC Institute of Molecular Oncology, Milan, Italy. SBC, APS, Argonne, Illinois, USA. SLS, PSI, Villigen, Switzerland. IDD, Branford, CT, USA. Sanwa Kagaku Kenkyusyo Ltd., Japan. E-mail: podjarny@igbmc.u-strasbg.fr