Tatiana Petrova

Factorizing Selectivity Determinants of Inhibitor Binding toward Aldose and Aldehyde Reductases: Structural and Thermodynamic Properties of the Aldose Reductase Mutant Leu300Pro−Fidarestat Complex

Structure of the Leu300Pro mutant of human aldose reductase (ALR2) in complex with the inhibitor fidarestat is determined. Comparison with the hALR2-fidarestat complex and the porcine aldehyde reductase (ALR1)-fidarestat complex indicates that the hydrogen bond between the Leu300 amino group of the wild-type and the exocyclic amide group of the inhibitor is the key determinant for the specificity of fidarestat for ALR2 over ALR1. Thermodynamic data also suggest an enthalpic contribution as the predominant difference in the binding energy between the aldose reductase mutant and the wild-type. An additional selectivity-determining feature is the difference in the interaction between the inhibitor and the side chain of Trp219, ordered in the present structure but disordered (corresponding Trp220) in the ALR1-fidarestat complex. Thus, the hydrogen bond ( approximately 7 kJ/mol) corresponds to a 23-fold difference in inhibitor potency while the differences in the interactions between Trp219(ALR2) and fidarestat and between Trp220(ALR1) and fidarestat can account for an additional 10-fold difference in potency.

X-ray-induced deterioration of disulfide bridges at atomic resolution.

Overall and site-specific X-ray-induced damage to porcine pancreatic elastase was studied at atomic resolution at temperatures of 100 and 15 K. The experiments confirmed that irradiation causes small movements of protein domains and bound water molecules in protein crystals. These structural changes occur not only at 100 K but also at temperatures as low as 15 K. An investigation of the deterioration of disulfide bridges demonstrated the following. (i) A decrease in the occupancy of S(γ) atoms and the appearance of new cysteine rotamers occur simultaneously. (ii) The occupancy decrease is observed for all S(γ) atoms, while new rotamers arise for some of the cysteine residues; the appearance of new conformations correlates with the accessibility to solvent. (iii) The sum of the occupancies of the initial and new conformations of a cysteine residue is approximately equal to the occupancy of the second cysteine residue in the bridge. (iv) The most pronounced changes occur at doses below 1.4 × 10(7) Gy, with only small changes occurring at higher doses. Comparison of the radiation-induced changes in an elastase crystal at 100 and 15 K suggested that the dose needed to induce a similar level of deterioration of the disulfide bonds and atomic displacements at 15 K to those seen at 100 K is more than two times higher.

X-Ray-Radiation-Induced Cooperative Atomic Movements in Protein

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is. We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to approximately 100 K and diffracting to 1 A. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.

Ultrahigh-resolution study of protein atomic displacement parameters at cryotemperatures obtained with a helium cryostat.

Two X-ray data sets for a complex of human aldose reductase (h-AR) with the inhibitor IDD 594 and the cofactor NADP(+) were collected from two different parts of the same crystal to a resolution of 0.81 A at 15 and 60 K using cold helium gas as cryogen. The contribution of temperature to the atomic B values was estimated by comparison of the independently refined models. It was found that although being slightly different for different kinds of atoms, the differences (deltaB) in the isotropic equivalents B of atomic displacement parameters (ADPs) were approximately constant (about 1.7 A(2)) for well ordered atoms as the temperature was increased from 15 to 60 K. The mean value of this difference varied according to the number of non-H atoms covalently bound to the parent atom. Atoms having a B value of higher than 8 A(2) at 15 K showed much larger deviations of deltaB from the average value, which might reflect partial occupancy of atomic sites. An analysis of the anisotropy of ADPs for individual atoms revealed an increase in the isotropy of ADPs with the increase of the temperature from 15 to 60 K. In a separate experiment, a 0.93 A resolution data set was collected from a different crystal of the same complex at 100 K using cold nitrogen as a cryogen. The effects of various errors on the atomic B values were estimated by comparison of the refined models and the temperature-dependent component was inferred. It was found that both decreasing the data redundancy and increasing the resolution cutoff led to an approximately constant increase in atomic B values for well ordered atoms.

Novel hexamerization motif is discovered in a conserved cytoplasmic protein from Salmonella typhimurium.

The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25xa0Å. The asymmetric unit contains a hexamer of structurally identical monomers. The monomer is a globular domain with a long β-hairpin protrusion that distinguishes this structure. This β-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexamer. We suggest that the structure of Stm3548 presents a new hexamerization motif. Because the residues participating in interdomain interactions are highly conserved among close members of protein family DUF1355 and buried solvent accessible area for the hexamer is significant, the hexamer is most likely conserved as well. A light scattering experiment confirmed the presence of hexamer in solution.

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