Structural studies on Pax-8 Prd domain/DNA complex.

Pax-8 is a member of the Pax family of transcription factors and is essential in the development of thyroid follicular cells. Pax-8 has two DNA-binding domains: the paired domain and the homeo domain. In this study, a preliminary X-ray diffraction analysis of the mammalian Pax-8 paired domain in complex with the C-site of the thyroglobulin promoter was achieved. The Pax-8 paired domain was crystallized by the hanging-drop vapor-diffusion method in complex with both a blunt-ended 26 bp DNA fragment and with a sticky-ended 24 bp DNA fragment with two additional overhanging bases. Crystallization experiments make clear that the growth of transparent crystals with large dimensions and regular shape is particularly influenced by ionic strength. The crystals of Pax-8 complex with blunt-ended and sticky-ended DNA, diffracted synchrotron radiation to 6.0 and 8.0 A resolution and belongs both to the C centered monoclinic system with cell dimensions: a = 89.88 A, b = 80.05 A, c = 67.73 A, and beta = 124.3 degrees and a = 256.56, b = 69.07, c = 99.32 A, and beta = 98.1 degrees , respectively. Fluorescence experiments suggest that the crystalline disorder, deduced by the poor diffraction, can be attributed to the low homogeneity of the protein-DNA sample. The theoretical comparative model of the Pax-8 paired domain complexed with the C-site of the thyroglobulin promoter shows the probable presence of some specific protein-DNA interactions already observed in other Pax proteins and the important role of the cysteine residues of PAI subdomain in the redox control of the DNA recognition.

HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations.

We demonstrate that in three cases of MC (two with immunocytoma), the IgM-RF+ component of their cryoprecipitated represents the circulating counterpart of the B-cell receptor (BCR) of the monoclonal overexpanded B-cell population. These IgMs were isolated and used to demonstrate a crossreactivity against both hepatitis C virus (HCV) NS3 antigen and the Fc portion of IgG. Epitopes were identified in a fraction of exemplary samples by using epitope excision approach (NS(31250-1334) and IgG Fc(345-355)). The same phenomenon of crossreactivity has been shown to occur in vivo after immunization of a mouse with the NS3(1251-1270) peptide. To verify if the same reaction was also present in MC samples characterized by an oligo/polyclonal B-cell proliferation, IgM crossreactivity was tested in 14 additional samples. Five out of the 14 were reactive against HCV NS3 and 11 out of 14 were reactive against IgG-Fc peptide. The data support the role of HCV NS3 antigen in a subset of patients with MC, whereas the high frequency of the IgG-Fc epitope suggests that these B cells originate from precursors strongly selected for auto-IgG specificity. We suggest that engagement of specific BCRs by NS3 (or NS3-immunocomplex) antigen could explain the prevalence of IgM cryoglobulins in these patients.

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase.

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.

Toward the de novo design of a catalytically active helix bundle: a substrate-accessible carboxylate-bridged dinuclear metal center.

De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complex of "due-ferri-1" (DF1), a protein patterned after the diiron-dimanganese class of redox-active proteins [Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298-6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13'), which prevent facile entry of metal ions and small molecules. We have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule, which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.

Crystallization and preliminary X-ray diffraction analysis of human transcobalamin, a vitamin B12-transporting protein.

Transcobalamin is a cobalamin-binding protein in mammalian plasma that facilitates the cellular uptake of vitamin B(12). Human transcobalamin was crystallized using polyethylene glycol and ethanol as precipitants. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.04, b = 145.27, c = 164.96 A. A complete data set to 3.2 A resolution was collected from a single crystal using synchrotron radiation. Estimation of the crystal packing (V(M) = 3.2 A(3) Da(-1)) and self-rotation function analysis suggest the presence of two molecules in the asymmetric unit related by non-crystallographic twofold symmetry.

Addition reactions of aldehydes to lithium enolates of 1,3-dioxolan-4-ones: a configurational reassessment

The results for the addition reactions of chiral lithium (2S)-enolates of 1,3-dioxolan-4-ones to aldehydes and to acetophenone, yielding the corresponding dioxolanone alcohols have been revised. The results reported herein differ from those reported in the literature, both in product distribution and in the stereochemical assignment of the products. In fact, in several cases no stereocontrol was observed at the C5 carbon atom of the lithium enolate. The (2S,5R,1'S)/(2S,5R,1'R) stereochemistry was also reassessed for several dioxolanone alcohols. The major conformers are considered to have an intramolecular hydrogen-bonded five-membered ring structure instead of the six-membered ring structure previously suggested for cyclic dioxolanone alcohols.

Crystallization and preliminary X-ray analysis of two pH-dependent forms of cytochrome c2 from Rhodopseudomonas palustris.

Cytochrome c(2) from Rhodopseudomonas palustris has been crystallized in two different crystal forms: a monoclinic form I at pH 4.4 from both reduced and oxidized protein solution and a trigonal form II at pH 9.0 from reduced protein solution. Complete 1. 7 and 1.4 A resolution data sets were collected from the oxidized form I and from the form II, respectively. The preliminary structures show an important change in the iron coordination environment in the trigonal form obtained at basic pH arising from the substitution of the Met ligand by an ammonia molecule.

Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins.

De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 A rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 A. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.

Similarities and differences between cobalamins and cobaloximes. Accurate structural determination of methylcobalamin and of LiCl- and KCl-containing cyanocobalamins by synchrotron radiation.

The accurate crystal structure determinations of MeCbl (1), CNCbl.2LiCl (2), and CNCbl.KCl (3), based on synchrotron diffraction data collected at 100 K and using high-quality single crystals, are reported. Refinements gave R1 indices of 0.0834 (1), 0.0434 (2), and 0.0773 (3). The influence of the water of crystallization and ion content on the crystal packing of these and other cobalamins (XCbl) is discussed, and a relationship between the crystal packing and the corrin side chain conformations is presented. An analysis of the bond lengths within the corrin moiety, based on 13 accurate structures with several X groups, shows that the trend of the C-C and C-N distances can be interpreted in terms of electronic and steric factors. The variation in structural, NMR and IR spectroscopic, and electrochemical properties are compared with those of cobaloximes, the B12 model, when X is varied. This comparison indicates that the pi-back-donation from metal to the CN axial ligand and the transmission of the trans influence of the X ligand are more effective in cobalamins than in cobaloximes. These findings are consistent with a significantly greater availability of electron charge on Co in cobalamins, and, hence, a semiquantitative evaluation of the electronic difference between the cobalt centers in the two systems is allowed.

Phosphorylase recognition and phosphorolysis of its oligosaccharide substrate: answers to a long outstanding question.

Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-alpha-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 A resolution; and the 2.1 A resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites -1 to +4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites -1 and +1, the site of catalysis, from the preferred conformation for alpha(1-4)-linked glucosyl polymers.