The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases.

BACKGROUND: . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS: . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS: . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.

The structure of the Escherichia coli phosphotransferase IIAmannitol reveals a novel fold with two conformations of the active site.

BACKGROUND: The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the cellular uptake and subsequent phosphorylation of carbohydrates. Moreover, the PTS plays a crucial role in the global regulation of various metabolic pathways. The PTS consists of two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II (EII). EIIs are usually composed of two cytoplasmic domains, IIA and IIB, and a transmembrane domain, IIC. The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydrate. Knowledge of the structures of the IIA proteins may provide insight into the mechanisms by which the PTS couples phosphorylation reactions with carbohydrate specificity. RESULTS: We have determined the crystal structure of the Escherichia coli mannitol-specific IIA domain, IIAmtl (M(r) 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIAmtl. The structure was refined at 1.8 A resolution to an R factor of 19.0% (Rfree 24.2%). The enzyme consists of a single five-stranded mixed beta sheet, flanked by helices on both sides. The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydrophobic residues. The sidechains of two conserved active-site residues, Arg49 and His111, adopt two different conformations in the four independent IIAmtl molecules. Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experiments, structural models of the HPr-IIAmtl complex were generated. CONCLUSIONS: The fold of IIAmtl is completely different from the structures of other IIA proteins determined so far. The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIAmtl is involved. A comparison of the HPr-IIAmtl model with models of HPr in complex with other IIA enzymes shows that the overall interaction mode between the two proteins is similar. Differences in the stabilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium.

Some physicochemical properties of bovine alpha s 2-casein.

The self-association of purified bovine alpha s 2-casein in 0.005 M EDTA, pH 6.7, at ionic strengths varying from 0.02 to 1.2 depends strongly on the ionic strength. The association reaches its maximum at an ionic strength (I) between 0.02 and 0.3 and can be described by the so-called isodesmic model. The standard free energy of association is about -38 kJ/mol at 20 degrees C. If it is assumed that the particles are spherical and have a voluminosity which is independent of the degree of association, then from the intrinsic viscosities at different ionic strengths the radius of the spherical particle (approximately equal to 3.7 nm), the voluminosity (approximately equal to 5 ml . g-1) and the solvation (approximately equal to 4.2. g . g-1) can be obtained.

Crystallization of enzyme IIB of the cellobiose-specific phosphotransferase system of Escherichia coli.

Crystals of enzyme IIB of the cellobiose-specific phosphotransferase system have been obtained from 15% polyethylene glycol 4000 using both streak-seeding and macroseeding techniques at 4 degrees. Crystals were grown with the hanging drop method of vapour diffusion. Addition of 2-propanol and benzamidine/HCl proved essential to obtain single crystals suitable for X-ray analysis. The crystals diffract to 1.8 A resolution and have the monoclinic space group P2(1), with cell dimensions a = 53.6 A, b = 31.7 A, c = 60.0 A and beta = 101.7 degrees. From a self-rotation function it seems likely that there are two molecules in the asymmetric unit related by a non-crystallographic 2-fold axis.

Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form.

The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.

The functional importance of structural differences between the mannitol-specific IIAmannitol and the regulatory IIAnitrogen.

The three-dimensional structures of the IIA domain of the mannitol-specific phosphoenol-pyruvate dependent phosphotransferase system (PTS) of Escherichia coli and the regulatory IIAntr enzyme have been compared. The enzymes are 20% identical in sequence and contain the sequence motif specific for IIA proteins belonging to the mannitol-fructose family of the PTS. The fold of the enzymes is nearly identical, which confirms their evolution from a common ancestor. Moreover, the phosphorylation site of IIAmtl (His65) and one of the observed conformations of the active site Arg49 are extremely similar to their equivalents (His73 and Arg57) in IIAntr. In contrast, His120, the equivalent of the second active site His111 of IIAmtl, is not located in the active site of IIAntr but points into the solvent on the other side of the molecule. The different position of His120 makes it unlikely that this residue assists in phosphoryl transfer in the regulatory IIA(ntr)s. As His120 is conserved in all IIAntr enzymes, it could have an essential role in the recognition of the target protein of IIAntr.

Crystal structure and assembly of a eukaryotic small heat shock protein.

The 2.7 A structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its alpha-crystallin domain and flanking extensions assemble into a dodecameric double disk. The folding of the monomer and assembly of the oligomer are mutually interdependent, involving strand exchange, helix swapping, loose knots and hinged extensions. In support of the chaperone mechanism, the substrate-bound dimers, in temperature-dependent equilibrium with higher assembly forms, have unfolded N-terminal arms and exposed conserved hydrophobic binding sites on the alpha-crystallin domain. The structure also provides a model by which members of the sHSP protein family bind unfolded substrates, which are involved in a variety of neurodegenerative diseases and cataract formation.

Crystal structure of eta-crystallin: adaptation of a class 1 aldehyde dehydrogenase for a new role in the eye lens.

Eta-crystallin is a retinal dehydrogenase that has acquired a role as a structural protein in the eye lens of elephant shrews, members of an ancient order of mammals. While it retains some activity toward retinal, which is oxidized to retinoic acid, the protein has acquired a number of specific sequence changes that have presumably been selected to enhance the lens role. The crystal structure of eta-crystallin, in common with class 1 and 2 ALDHs, is a dimer of dimers. It has a better-defined NAD binding site than those of related mammalian ALDH1 enzymes with the cofactor bound in the "hydride transfer" position in all four monomers with small differences about the dimer dyads. Although the active site is well conserved, the substrate-binding site is larger in eta-crystallin, and there are some mutations to the substrate access tunnel that might affect binding or release of substrate and product. It is possible that eta-crystallin has lost flexibility to improve its role in the lens. Enhanced binding of cofactor could enable it to act as a UV/blue light filter in the lens, improving visual acuity. The structure not only gives a view of a "natural mutant" of ALDH1 illustrating the adaptive conflict that can arise in multifunctional proteins, but also provides a well-ordered NAD binding site structure for this class of enzymes with important roles in development and health.