Antonio Párraga

The structure of plasmid‐encoded transcriptional repressor CopG unliganded and bound to its operator

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon–helix–helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13‐bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19‐bp oligonucleotide, containing the pseudosymmetric element, with two β‐ribbons inserted into the major groove. The DNA is bent 60° by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix–turn–helix regions of unrelated DNA‐binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon–helix–helix and helix–turn–helix proteins.

Structure of human biliverdin IXβ reductase, an early fetal bilirubin IXβ producing enzyme

Biliverdin IXβ reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXβ, the major heme catabolite during early fetal development. BVR-B displays a preference for biliverdin isomers without propionates straddling the C10 position, in contrast to biliverdin IXα reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP+ at 1.15 Å resolution. Human BVR-B is a monomer displaying an α/β dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IVα, biliverdin IXα, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates and inhibitors bind primarily through hydrophobic interactions, explaining its broad specificity. The reducible atom of both biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the biliverdin IXα isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzymes preference for NADP(H) and its B-face stereospecificity.

The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme-GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

Purification, crystallization and preliminary X‐ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double‐stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu‐C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino‐ and carboxy‐terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P42212 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X‐ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two‐dimensional crystals analyzed previously by electron microscopy.

On the reaction mechanism of class Pi glutathione S-transferase.

Theoretical calculations were performed to examine the ionization of the phenolic group of Tyr7 and the thiol group of glutathione in aqueous solution and in the protein class‐pi glutathione S‐transferase (GST‐Pi). Three model systems were considered for simulations in the protein environment: the free enzyme, the complex between glutathione and the enzyme, and the complex between 1‐chloro‐2.4‐dinitrobenzene, glutathione, and the enzyme. The structures derived from Molecular Dynamics simulations were compared with the crystallographic data available for the complex between the inhibitor S‐(p‐nitrobenzyl)glutathione and GST‐Pi, the glutathione‐bound form of GST‐Pi, and the free enzyme carboxymethylated in Cys47. Free‐energy perturbation techniques were used to determine the thermodynamics quantities for ionization of the phenol and thiol groups. The functional implications of Tyr7 in the activation of the glutathione thiol group are discussed in the light of present results, which in agreement with previous studies suggest that Tyr7 in un‐ionized form contributes to the catalytic process of glutathione S‐transferase, the thiolate anion being stabilized by hydrogen bond with Tyr7 and by interactions with hydrating water molecules. Proteins 28:530–542, 1997

Detection of elsamicin-DNA binding specificity by restriction enzyme cleavage

The sequence specificity of elsamicin A, an anti‐tumour antibiotic, binding to DNA was elucidated considering the inhibition of the rate of digestion of linearised pBR322 DNA by AnII, ClaI, EcoRI, HindIII and NruI restriction enzymes. Elsamicin A inhibits the rate of digestion by NruI (recognition sequence TCG/CGA) to a greater extent than it does for the other enzymes thus evidencing the sequence‐selective binding of elsamicin to CGC regions in DNA. Our results also show the important role of the neighbouring sequences in the elsamicin A‐DNA interactions and their effects on the cleavage by restriction enzymes.

Shaped protein single crystals

The formation of protein single crystals grown with the shape controlled by the geometry of the capillary used as a growth cell is presented. The shaped crystals show strong birefringence under crossed nicols and diffract as single crystals up to 1.74 A.

Dichlorobis(pentachlorophenyl) germane and derivatives; synthesis of bis(pentachlorophenyl) germanediol and molecular structure of diiodobis(pentachlorophenyl) germane

Abstract Dichlorobis(pentachlorophenyl)germane (3) has been prepared by reaction of GeCl4 with either pentachlorophenyllithium or pentachlorophenylmagnesium chloride. After neutral hydrolysis of 3, bis(pentachlorophenyl)germanediol (8) has been isolated and characterized as a stable solid. Thermal dehydration of 8 has been studied by thermogravimetry (TG) and differential scanning calorimetry (DSC), the product being {(C6Cl5)2GeOn}. Reaction of 3 with methanol leads to dimethoxybis(pentachlorophenyl)germane (4). Reduction of 3 with LiAlH4 gives bis(pentachlorophenyl)germane (5) which, on treatment with Br2 and I2, gives dibromo- (6) and diiodobis(pentachlorophenyl)germane (7), respectively. The molecular structure of the diiodogermane 7 has been fully established by X-ray crystallography.