Core Β-sheet Research Articles

α → β transition of β-lactoglobulin as evidenced by heteronuclear NMR

Whereas bovine β-lactoglobulin is a predominantly β-sheet protein, it has a marked α-helical preference and can be considered to be a useful model of the α → β transition, a key issue for understanding the folding and biological function of a number of proteins. In order to understand the mechanism of the α → β transition, the backbone structures of the recombinant bovine β-lactoglobulin A in the native state and in the highly helical state induced by 2,2,2-trifluoroethanol were characterized by 1H, 13C and 15N multidimensional NMR spectroscopy. Overall, the secondary structures in the native state were similar to those of the crystal structure. On the other hand, β-lactoglobulin in the 2,2,2-trifluoroethanol state was composed of many α-helical segments. The presence of the persistent α-helices in the helical state and the core β-sheet in the native state suggested that during folding native-like core β-sheet and several non-native helices are formed first and the remaining β-sheet is subsequently “induced” through interaction with the pre-existing β-sheet.

Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases.

The crystal structure of the recombinant fructose-2,6-bisphosphatase domain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was determined by multiwavelength anomalous dispersion phasing and refined at 2.5 A resolution. The selenomethionine-substituted protein was induced in the methionine auxotroph, Escherichia coli DL41DE3, purified, and crystallized in a manner similar to that of the native protein. Phase information was calculated using the multiwavelength anomalous dispersion data collected at the X-ray wavelengths near the absorption edge of the K-shell alpha electrons of selenium. The fructose-2,6-bisphosphatase domain has a core alpha/beta structure which consists of six stacked beta-strands, four parallel and two antiparallel. The core beta-sheet is surrounded by nine alpha-helices. The catalytic site, as defined by a bound phosphate ion, is positioned near the C-terminal end of the beta-sheet and close to the N-terminal end of an alpha-helix. The active site pocket is funnel-shaped. The narrow opening of the funnel is wide enough for a water molecule to pass. The key catalytic residues, including His7, His141, and Glu76, are near each other at the active site and probably function as general acids and/or bases during a catalytic cycle. The inorganic phosphate molecule is bound to an anion trap formed by Arg6, His7, Arg56, and His141. The core structure of the Fru-2,6-P2ase is similar to that of the yeast phosphoglycerate mutase and the rat prostatic acid phosphatase. However, the structure of one of the loops near the active site is completely different from the other family members, perhaps reflecting functional differences and the nanomolar range affinity of Fru-2,6-P2ase for its substrate. The imidazole rings of the two key catalytic residues, His7 and His141, are not parallel as in the yeast phosphoglycerate mutase. The crystal structure is used to interpret the existing chemical data already available for the bisphosphatase domain. In addition, the crystal structure is compared with two other proteins that belong to the histidine phosphatase family.

The Monofunctional Chorismate Mutase from Bacillus subtilis: Structure Determination of Chorismate Mutase and Its Complexes with a Transition State Analog and Prephenate, and Implications for the Mechanism of the Enzymatic Reaction

Structures have been determined for chorismate mutase from Bacillus subtilis and of complexes of this enzyme with product and an endo -oxabicyclic transition state analog using multiple isomorphous replacement plus partial structure phase combination and non-crystallographic averaging. In addition to 522 water molecules, the model includes 1380 of the 1524 amino acid residues of the four trimers (each containing 3 × 127 amino acid residues) in the asymmetric unit. Refinement to 1·9 Å resolution yields 0·194 for R and r.m.s. deviations from ideal values of 0·014 Å for bond lengths and 2·92° for bond angles. The trimer resembles a β-barrel structure in which a core β-sheet is surrounding by helices.The structures of the two complexes locate the active sites which are at the interfaces of adjacent pairs of monomers in the trimer. These structures have been refined a 2·2 Å to a crystallographic R value of 0·18 and show r.m.s. deviations from ideal values of 0·013 Å for bond lengths and 2·84° or 3·05° for bond angles, respectively. The final models have 1398 amino acid residues, nine prephenate molecules and 503 water molecules in the product complex, and 1403 amino acid residues, 12 inhibitor molecules and 530 water molecules in the transition state complex.The active sites of all three of these structures are very similar and provide a structural basis for the biochemical studies that indicate a pericyclic mechanism for conversion of chorismate to prephenate. The absence of reactive catalytic residues on the enzyme, the selective binding of the single reactive conformation of chorismate, the stabilization of the polar transition state, and the possible role of the C-terminal region in "capping" the active site are factors which relate these structures to the million-fold rate enhancement of this reaction.