An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apoliporprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) With cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal from were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps. which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal Terms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this anti-parallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.

The XRCC1 DNA repair protein contains two regions of approximately 100 amino acids each that share homology with the BRCT (BRCA1 carboxyl terminus) domain superfamily. These two regions of XRCC1 have been shown to interact independently with DNA ligase III and poly(ADP-ribose)polymerase as part of a mechanism involved in the repair of DNA single-strand breaks. To understand how these BRCT regions specify protein-protein interactions and contribute to DNA repair function, we have overexpressed and purified the distal BRCT domain of XRCC1 with the goal of structure determination. The cDNA encoding this BRCT region (X1BRCTb) was inserted into the pET29 bacterial expression vector; the polypeptide was expressed in mostly soluble form and then purified by anion-exchange and gel filtration chromatography. Crystallization screening with the purified material resulted in the formation of large bipyramidal crystals. Crystals formed within several hours at room temperature from salt solutions of ammonium sulfate. Crystals diffract to similar to 2.85 Angstrom and were found to be in space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2) with unit cell dimensions a = 100.43 Angstrom, c = 105.62 Angstrom. Crystals of similar character have also been obtained after incorporation of selenomethionine during expression of the protein. Efforts are now under way to determine the molecular structure of-the X1BRCTb domain. These studies are likely to give insight into the interaction between XRCC1 and DNA Ligase III and into general structural features of BRCT domains that exist in many other proteins.

We investigated the binding of ATP in the presence and absence of Mg2+ to dimeric muscle creatine kinase (CK) by isothermal titration microcalorimetry as a function of pH and temperature. The thermodynamic parameters for these events show that (1) binding of nucleotide to the CK active site does not involve proton exchange with the buffer and (2) the active sites are the only nucleotide binding sites on CK. Interdependence of the active sites in the dimer could not be demonstrated. As CK undergoes major structural changes upon Mg-nucleotide binding, a thermodynamic cycle was employed to calculate the contributions of domain movements to the observed enthalpies.

Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.

We show that the mutation of an uncharged residue far from the active site to another uncharged residue can have effects on the active site without disturbing the overall structure of the protein. Cis-proline 207 of mitochondrial creatine kinase was mutated to alanine. The mutant showed a decrease in the pH-optimum for ATP synthesis by 1.5 units while the maximum relative activity was lowered to 53% of the wild-type enzyme. In the direction of ATP consumption, the pH optimum was lowered by 1.3 units and the maximum relative activity was 49% of the wild-type enzyme. The enzyme kinetic parameters Km and Kd for the substrates did not change dramatically, indicating a largely unperturbed active site. Small-angle X-ray scattering was used to investigate the structural change concomitant with the mutation, yielding a scattering profile only slightly different from that of the wild-type enzyme. Neither the radius of gyration nor the molecular mass showed any significant differences, leading to the conclusion that quarternary organization and fold of the mutant and the wild-type enzymes were similar. Theoretical analysis suggests the most probable primary source of structural change to be a transition of residue 207 peptide bond torsional angle co from the cis to the trans configuration.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

A histidine residue with a pK(a) of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mi(b)-CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small-angle X-ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters K-m or V-max, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.

Small-angle X-ray and neutron scattering have been used to investigate structural changes upon binding of individual substrates or a transition state analogue complex (TSAC), consisting of Mg-ADP, creatine and KNO3 to creatine kinase isoenzymes (dimeric M-CK and octameric Mi-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-ATP and TSAC, whereas creatine alone had only a small effect. In Mi-CK, the radius of gyration was reduced from 55.6 Å (free enzyme) to 48.9 Å (enzyme + Mg-ATP) and to 48.2 Å (enzyme + TSAC). The experiments performed with M-CK showed similar changes from 28.0 Å (free enzyme) to 25.6 Å (enzyme + Mg-ATP) and to 25.5 Å (enzyme + TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK showed the same behaviour: a change of the radius of gyration from 21.5 Å (free enzyme) to 19.7 Å (enzyme + MG-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a magnesium-nucleotide induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In creatine kinase, however, further movements must be involved in the large conformational change.

Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is one of the four enzymes of the peroxisomal β-oxidation pathway. Here, we report the full-length human cDNA sequence and the localization of the corresponding gene on chromosome 3q26.3-3q28. The cDNA sequence spans 3779 nucleotides with an open reading frame of 2169 nucleotides. The tripeptide SKL at the carboxy terminus, known to serve as a peroxisomal targeting signal, is present. DNA sequence comparison of the coding region showed an 80% homology between human and rat bifunctional enzyme cDNA. The 3′ noncoding sequence contains 117 nucleotides homologous to an Alu repeat. Based on sequence comparison, we propose that these nucleotides are a free left Alu arm with 86% homology to the Alu-J family. RNA analysis shows one band with highest intensity in liver and kidney. This cDNA will allow in-depth studies of molecular defects in patients with defective peroxisomal bifunctional enzyme. Moreover, it will also provide a means for studying the regulation of peroxisomal β-oxidation in humans.

Tau protein is a member of the family of microtubule-associated proteins, which support microtubule polymerization and stability. Under pathological conditions, tau is a major constituent of neurofibrillary tangles in nerve cells of patients with Alzheimer's disease. Neurofibrillary tangles share some morphological, biochemical and immunological properties with cytoplasmic inclusions associated with other diseases, such as Mallory bodies in the livers of patients with alcoholic hepatitis and in corresponding mouse models. Recently a Mallory body component was identified that in molecular mass and isoelectric point resembles the abnormally phosphorylated tau of neurofibrillary tangles. There has been, however, so far no report describing the occurrence of tau in normal liver. We now demonstrate the expression of two tau isoforms containing three and four repeats, respectively, of the microtubule binding domains in normal mouse liver and kidney. This finding provides evidence for a physiological role of tau in the liver and, consequently, the basis for the involvement of tau in pathological situations.

Tau protein is a member of the family of microtubule-associated proteins, which support microtubule polymerization and stability. Under pathological conditions, tau is a major constituent of neurofibrillary tangles in nerve cells of patients with Alzheimer's disease. Neurofibrillary tangles share some morphological, biochemical and immunological properties with cytoplasmic inclusions associated with other diseases, such as Mallory bodies in the livers of patients with alcoholic hepatitis and in corresponding mouse models. Recently a Mallory body component was identified that in molecular mass and isoelectric point resembles the abnormally phosphorylated tau of neurofibrillary tangles. There has been, however, so far no report describing the occurrence of tau in normal liver. We now demonstrate the expression of two tau isoforms containing three and four repeats, respectively, of the microtubule-binding domains in normal mouse liver and kidney. This finding provides evidence for a physiological role of tau in the liver and, consequently, the basis for the involvement of tau in pathological situations.