Preliminary crystallographic studies on bovine lactoferrin

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.     

Preliminary crystallographic studies of copper(II)- and oxalate-substituted human lactoferrin

As part of a comparative study on the binding of different metals and anions by human lactoferrin, we have prepared and crystallized: (1) dicupric lactoferrin with Cu2+ and carbonate in each site (Cu2Lf); and (2) a lactoferrin complex with Cu2+ and carbonate in one site, and Cu2+ and oxalate in the other (Cu2oxLf). Crystals of Cu2Lf are orthorhombic: a = 155.9, b = 97.0, c = 56.0 A, space-group P2(1)2(1)2(1); those of Cu2oxLf are also orthorhombici a = 155.9, b = 97.1, c = 56.2 A, space-group P2(1)2(1)2(1). Both are isomorphous with diferric human lactoferrin, Fe2Lf. Diffractometer data to 2.6 A and 2.5 A have been collected for Cu2Lf and Cu2oxLf, respectively. Difference maps show that the main effect of substitution of Cu2+ for Fe3+ is a small shift (0.5 to 1.0 A) in the metal position in each site. For Cu2oxLf the oxalate ion is found to be accommodated in the C-lobe, bound to copper in a bidentate mode, causing only small local changes, in the positions of adjacent Arg and Tyr side-chains.     

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.     

Cancer prevention by bovine lactoferrin: from animal studies to human trial

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and, despite improved colonoscopic screening, CRC is a leading cause of death from cancer. Administration of bovine lactoferrin (bLF) suppresses carcinogenesis in the colon and other organs of test animals, and recently it was shown that ingestion of bLF inhibits the growth of adenomatous polyps in human patients. Here we review work which established bLF as an anti-carcinogenic agent in laboratory animals and the results of a clinical trial which demonstrated that bLF can reduce the risk of colon carcinogenesis in humans.     

Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.     

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.     

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.     

Anion binding properties of human serum albumin from halide ion quadrupole relaxation.

The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more useful as a probe for the specific anion binding sites than the other two halide ions. The findings with human serum albumin in this and other respects are probably of general relevance in studies of protein-anion interactions. In addition to competition experiments, the magnitude of the relaxation rate is also discussed. Line broadening not related to anion binding to the protein is found to be small. A comparison of transverse and longitudinal 35Cl relaxation rates gives a value for the quadrupole coupling constant of the high affinity sites in good agreement with a calculated coupling constant assuming anion binding to arginine.     

The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors.

The oligosaccharides of human lactoferrin were enzymatically removed with glycopeptidase F, resulting in a preparation containing partial and fully deglycosylated human lactoferrin. The derivatives were separated by Concanavalin A affinity chromatography and compared with native human lactoferrin with respect to their ability to bind to bacterial receptors. Competitive binding experiments demonstrated that the lactoferrin derivatives were equally capable as native lactoferrin in binding to receptors of Neisseria meningitidis, Neisseria gonorrhoeae, and Moraxella catarrhalis. This result indicates that the oligosaccharides on human lactoferrin are not essential for binding to the bacterial receptors.     

Kinetic and crystallographic studies of glucopyranosylidene spirothiohydantoin binding to glycogen phosphorylase B.

Glucopyranosylidene spirothiohydantoin (TH) has been identified as a potential inhibitor of both muscle and liver glycogen phosphorylase b (GPb) and a (GPa) and shown to diminish liver GPa activity in vitro. Kinetic experiments reported here show that TH inhibits muscle GPb competitively with respect to both substrates phosphate (K(i)=2.3 microM) and glycogen (K(i)=2.8 microM). The structure of the GPb-TH complex has been determined at a resolution of 2.26 A and refined to a crystallographic R value of 0.193 (R(free)=0.211). The structure of GPb-TH complex reveals that the inhibitor can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provides a basis of understanding potency and specificity of the inhibitor. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the glucose complex, while the rigid thiohydantoin group is in a favourable electrostatic environment and makes additional polar contacts to the protein.     

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg(2+), and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the alpha3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg(2+). The new structure provides a plausible mechanism for the catalysis of UPPs.     

The compatible-solute-binding protein OpuAC from Bacillus subtilis: ligand binding, site-directed mutagenesis, and crystallographic studies

In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-A resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.     

X-ray crystallographic studies of streptavidin mutants binding to biotin

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.     

A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.     

Oxysterols: biological activities and physicochemical studies.

To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3 beta-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.