Hugo L. Monaco

The Transthyretin—Retinol-Binding Protein Complex

Transthyretin (TTR, formerly called prealbumin), one of the transporters of the hormone thyroxine and the lipocalin retinol-binding protein (RBP), the specific carrier of the vitamin, are known to form, under physiological conditions, a macromolecular complex that is believed to play an important physiological role: prevention of glomerular filtration of the low molecular weight RBP in the kidneys. The physiological significance of complex formation is discussed first, followed by a brief description of the three-dimensional structure of the two participating proteins. The two X-ray models of the complex available are subsequently discussed and compared and finally the non-crystallographic evidence that supports these models is reviewed.

Crystal structure of chicken riboflavin-binding protein

The crystal structure of chicken egg white riboflavin‐binding protein, determined to a resolution of 2.5 Å, is the prototype of a family that includes other riboflavin‐ and folate‐binding proteins. An unusual characteristic of these molecules is their high degree of cross‐linking by disulfide bridges and, in the case of the avian proteins, the presence of stretches of highly phosphorylated polypeptide chain. The structure of chicken egg white riboflavin‐binding protein is characterized by a ligand‐binding domain and a phosphorylated motif. The ligand‐binding domain has a fold that appears to be strongly conditioned by the presence of the disulfide bridges. The phosphorylated motif, essential for vitamin uptake, is made up of two helices found before and after the flexible phosphorylated region. The riboflavin molecule binds to the protein with the isoalloxazine ring stacked in between the rings of Tyr75 and Trp156. This geometry and the proximity of other tryptophans explain the fluorescent quenching observed when riboflavin binds to the protein.

The Antineoplastic Lectin of the Common Edible Mushroom (Agaricus bisporus) Has Two Binding Sites, Each Specific for a Different Configuration at a Single Epimeric Hydroxyl

The lectin from the common mushroom Agaricus bisporus, the most popular edible species in Western countries, has potent antiproliferative effects on human epithelial cancer cells, without any apparent cytotoxicity. This property confers to it an important therapeutic potential as an antineoplastic agent. The three-dimensional structure of the lectin was determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two β sheets connected by a helix-loop-helix motif. Selectivity was studied by examining the binding of four monosaccharides and seven disaccharides in two different crystal forms. The T-antigen disaccharide, Galβ1–3GalNAc, mediator of the antiproliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The binding of N-acetylgalactosamine overlaps with that moiety of the T antigen, but surprisingly, N-acetylglucosamine, which differs from N-acetylgalactosamine only in the configuration of epimeric hydroxyl 4, binds at a totally different site on the opposite side of the helix-loop-helix motif. The lectin thus has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl. The structure of the protein and its two carbohydrate-binding sites are described in detail in this study.

Partially folded structure of monomeric bovine β-lactoglobulin

Bovine β‐LG (β‐lactoglobulin) has been studied under a variety of solution conditions by one‐ and two‐dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured β‐sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of β‐LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.

Identification of a conserved hydrophobic cluster in partially folded bovine β-lactoglobulin at pH 2

BACKGROUND NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions formed during folding. Although the complete structural characterization of partially folded proteins is a very difficult task, the identification of structured subsets, such as hydrophobic clusters, is of value in understanding the structural organization of such states. Here, we report the NMR characterization, in acidic conditions (pH 2), of a well-defined hydrophobic cluster localized in the core of bovine beta-lactoglobulin. RESULTS The existence of a small hydrophobic cluster present in the lipocalin protein family has been assessed on the basis of structural alignment and NRM data obtained for the partially folded bovine beta-lactoglobulin. The presence of the cluster had been predicted identifying those residues that are highly conserved in most members of the family. An NMR study conducted at pH 2, where the protein exhibits a very stable beta-core together with disordered regions, reveals the presence of NOEs among sidechains of 11 hydrophobic residues centered around Trp19 and pointing towards the interior of the protein. This buried cluster is found to be unusually stable at pH 2, not only at room temperature but also at 323K. Furthermore, conserved hydrophobic residues pointing towards the surface of the protein define a hydrophobic surface patch located in a groove between the strands and the helix. CONCLUSIONS The detected buried cluster most likely plays an important role in bovine beta-lactoglobulin stability. The analysis of five structurally related proteins reveals that the same extended cluster is present in these structures. We propose that the buried cluster may represent the internal binding site as well and that the hydrophobic surface patch is involved in a second external binding site.

Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 Å resolution

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 Å resolution by X-ray crystallography. Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built. This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32. The protein is structured as a compact 10-stranded-β-barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule. The NH2-terminus portion of the molecule contains two short α-helices. The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods.

The primary structure of a basic (pI 9.0) fatty acid-binding protein from liver of Gallus domesticus

The complete amino acid sequence of a basic (pI 9.0) fatty acid-binding protein purified from liver of Gallus domesticus was determined by automated Edman degradation of tryptic, CNBr/HFBA and Staphylococcus aureus protease peptides. The protein contains 125 amino acid residues which correspond to a molecular mass of 14094. The identification of the blocked N-terminus Ac-Ala required digestion of a SV-8 peptide with the acylamino acid-releasing enzyme prior to sequence analysis. Sequence comparison shows that chicken liver basic-FABP has a significant similarity to other proteins belonging to the superfamily of intracellular lipid molecule binding proteins. Moreover, these sequence data confirm that basic-FABP probably binds its substrate in a slightly different way when compared with other FABPs. Basic-FABP was submitted to the EMBL Data Library with an accession number of P80226.

Structure and properties of the C-terminal domain of insulin-like growth factor-binding protein-1 isolated from human amniotic fluid.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Å resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe2+ ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

Structural and biochemical characterization of a new type of lectin isolated from carp eggs

A previously unidentified glycoprotein present in the eggs of the carp ( Cyprinus carpio ) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N -acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N -acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp ( Carassius auratus gibelio ) and 78% identity with a gene in the cDNA library of the zebrafish ( Danio rerio ).

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.