No abstract

The crystal structure of mitochondrial malate dehydrogenase from porcine heart contains four identical subunits in the asymmetric unit of a monoclinic cell. Although the molecule functions as a dimer in solution, it exists as a tetramer with 222 point symmetry in the crystal. The crystallographic refinement was facilitated in the early stages by using weak symmetry restraints and molecular dynamics. The R-factor including X-ray data to 1.83-A resolution was 21.1%. The final root mean square deviation from canonical values is 0.015 A for bond lengths and 3.2 degrees for bond angles. The resulting model of the tetramer includes independent coordinates for each of the four subunits allowing an internal check on the accuracy of the model. The crystalline mitochondrial malate dehydrogenase tetramer has been analyzed to determine the surface areas lost at different subunit-subunit interfaces. The results show that the interface with the largest surface area is the same one found in cytosolic malate dehydrogenase. Each of the subunits contains a bound citrate molecule in the active site permitting the elaboration of a model for substrate binding which agrees with that found for the crystalline enzyme from Escherichia coli. The environment of the N-terminal region of the crystallographic model has been studied because the functional protein is produced from a precursor. This precursor form has an additional 24 residues which are involved in mitochondrial targeting and, possibly, translocation. The crystallographic model of mitochondrial malate dehydrogenase has been compared with its cytosolic counterpart from porcine heart and two prokaryotic enzymes. Small but significant differences have been found in the polar versus nonpolar accessible surface areas between the mitochondrial and cytosolic enzymes. Using least squares methods, four different malate dehydrogenases have been superimposed and their consensus structure has been determined. An amino acid sequence alignment based on the crystallographic structures describes all the conserved positions. The consensus active site of these dicarboxylic acid dehydrogenases is derived from the least squares comparison.

In the present study, the synthesis of the 5.5.6. and 5.6.5. spiro bicyclic lactam PLG peptidomimetics, compounds 3 and 4, respectively, was undertaken. These peptidomimetics were designed to examine the following: (1) the effect that changing the size of the thiazolidine and lactam ring systems would have on the ability of these systems to mimic the type-II beta-turn and (2) the effect that these structural perturbations would have on the ability of the peptidomimetics to modulate dopamine receptors. Through the use of the [3H]spiroperidol/N-propylnorapomorphine (NPA) dopamine D2 receptor competitive binding assay, 3 and 4, at a concentration of 100 nM, decreased the dissociation constant of the high-affinity state of the dopamine receptor for the agonist. These effects were observed when either Gpp(NH)p was absent or present and they were comparable to those produced by PLG at a concentration of 1 microM. Peptidomimetics 3 and 4 also increased the percentage of D2 receptors that existed in the high-affinity state. Even with Gpp(NH)p present, 3 and 4 were able to return the RH/RL ratios to values observed in the respective controls where Gpp(NH)p was absent. Furthermore, both peptidomimetics were able to attenuate the Gpp(NH)p-induced shift to the low-affinity state to a greater extent than PLG. Peptidomimetics 3 and 4 were evaluated in vivo as modulators of apomorphine-induced rotational behavior in the 6-hydroxydopamine-lesioned rat model of hemiparkinsonism, and each affected the rotational behavior in a bell-shaped dose-response relationship producing increases of 95 +/- 31% (0.01 mg/kg, ip) and 88 +/- 14% (0.001 mg/kg, ip), respectively. In comparison, the previously reported 5.5.5. spiro bicyclic lactam 2 increased rotational behavior by 25 +/- 11% (0.01 mg/kg, ip).

The X-ray crystal structures of six salts composed of amino acids and sulfonated azo dyes have been determined, four of them at low temperature (173 K). The compounds are dl-lysine/4-[(4-hydroxyphenyl)azo]benzenesulfonate (HABS) monohydrate (1), dl-lysine/7-hydroxy-8-(phenylazo)-1,3-naphthalenedisulfonate (Orange G) dihydrate (2), l-lysine/Orange G 1.5-hydrate (3), dl-histidine/Orange G trihydrate (4), l-histidine/Orange G trihydrate (5), and tosylarginine methyl ester (TAME)/4-[(2-hydroxy-6-tert-butyl-1-naphthalenyl)azo]benzenesulfonate (“Little Rock Orange,” LRO) (6). By virtue of their basic side chains, these amino acids are the ones most important in the binding interactions between proteins and sulfated macromolecules such as glycosaminoglycans in living systems. The sulfonate salts described here serve as model systems for these interactions. Close intermolecular approaches between the dye sulfonate groups and neighboring amino acids and water molecules are examined, and the graph-set formalism is used to describe packing patterns and to identify corresponding interactions in different crystal structures. The recurrence of certain interactions between sulfonate groups and amino acid functional groups in these small-molecule crystal structures, including numerous interactions mediated by water molecules, suggests specificity that may also be a feature of the interactions between proteins and sulfated biological macromolecules.