The formation of a flexible DNA-binding protein chain is required for efficient DNA unwinding and adenovirus DNA chain elongation.

The adenovirus DNA-binding protein (DBP) binds cooperatively to single-stranded DNA (ssDNA) and stimulates both initiation and elongation of DNA replication. DBP consists of a globular core domain and a C-terminal arm that hooks onto a neighboring DBP molecule to form a stable protein chain with the DNA bound to the internal surface of the chain. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. As shown by x-ray diffraction of different crystal forms of the C-terminal domain, the C-terminal arm can adopt different conformations, leading to flexibility in the protein chain. This flexibility is a function of the hinge region, the part of the protein joining the C-terminal arm to the protein core. To investigate the function of the flexibility, proline residues were introduced in the hinge region, and the proteins were purified to homogeneity after baculovirus expression. The mutant proteins were still able to bind ss- and double-stranded DNA with approximately the same affinity as wild type, and the binding to ssDNA was found to be cooperative. All mutant proteins were able to stimulate the initiation of DNA replication to near wild type levels. However, the proline mutants could not support elongation of DNA replication efficiently. Even the elongation up to 26 nucleotides was severely impaired. This defect was also seen when DNA unwinding was studied. Binding studies of DBP to homo-oligonucleotides showed an inability of the proline mutants to bind to poly(dA)(40), indicating an inability to adapt to specific DNA conformations. Our data suggest that the flexibility of the protein chain formed by DBP is important in binding and unwinding of DNA during adenovirus DNA replication. A model explaining the need for flexibility of the C-terminal arm is proposed.

Structure of dimeric and monomeric erabutoxin a refined at 1.5 A resolution.

Erabutoxin a has been crystallized in its monomeric and dimeric forms. The structures were refined at 1.50 and 1.49 A resolution, respectively, using synchrotron radiation data. The crystals belong to space group P212121, with cell dimensions a = 49.84, b = 46.62, c = 21.22 A for the monomer and a = 55.32, b = 53.54, c = 40.76 A for the dimer. Using starting models from earlier structure determinations, the monomeric structure refined to an R value of 16.7% (8004 unique reflections, 17.0-1.50 A resolution range), while the dimeric structure has been solved by the molecular-replacement method with a final R value of 16.9% (19 444 unique reflections, 17.4-1.49 A resolution range). The high-resolution electron-density maps clearly revealed significant discrete disorder in the proteins and allowed an accurate determination of the solvent structure. For the monomer, the side chains of six residues were modelled with alternate conformers and 106 sites for water molecules and one site for a sulfate ion were included in the final model, whereas for the dimer, 206 sites for water molecules were included and both C-terminal residues together with the side chains of 11 residues adopted alternative conformations. A comparison was made with earlier structure determinations. The features of the solvent structure of the erabutoxin molecules are discussed in detail.

ATP-independent DNA unwinding by the adenovirus single-stranded DNA binding protein requires a flexible DNA binding loop.

The adenovirus DNA binding protein (DBP) binds cooperatively to single-stranded (ss) DNA and stimulates both initiation and elongation of DNA replication. DBP forms protein filaments via a C-terminal arm that hooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Another conserved part of DBP forms an unstructured flexible loop that is probably directly involved in contacting DNA. By making appropriate deletion mutants that do not distort the overall DBP structure, the influence of the C-terminal arm and the flexible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface plasmon resonance we show that both parts of the protein are required for high affinity binding. Deletion of the C-terminal arm leads to an extremely labile DBP-ssDNA complex indicating the importance of multimerization. The flexible loop is also required for optimal stability of the DBP-ssDNA complex, providing additional evidence that this region forms part of the ssDNA-binding surface of DBP. Both deletion mutants are still able to stimulate initiation of DNA replication but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Surprisingly, mixtures containing both mutants do stimulate elongation. Mixing the purified mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These results provide evidence that the C-terminal arm and the flexible loop have distinct functions in unwinding during replication. We propose the following model for ATP-independent DNA unwinding by DBP. Multimerization via the C-terminal arm is required for the formation of a protein filament that saturates the displaced strand. A high affinity of a DBP monomer for ssDNA and subsequent local destabilization of the replication fork requires the flexible loop.

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis.

In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.

The crystal structure of the complex of concanavalin A with 4'-methylumbelliferyl-alpha-D-glucopyranoside.

Concanavalin A (Con A) is the best known plant lectin, with important biological properties arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complex of Con A with 4'-methylumbelliferyl-alpha-D-glucopyranoside (alpha-MUG) has been crystallized in space group P2(1) with cell dimensions a = 81.62 A, b = 128.71 A, c = 82.23 A, and beta = 118.47 degrees. X-ray diffraction intensities to 2.78 A have been collected. The structure of the complex was solved by molecular replacement and refined by simulated annealing methods to a crystallographic R-factor value of 0.182 and a free-R-factor value of 0.216. The asymmetric unit contains four subunits arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site at the surface of each subunit, with the nonsugar (aglycon) part adopting a different orientation in each subunit. The aglycon orientation, although probably determined by packing of tetramers in the crystal lattice, helps to characterize the orientation of the saccharide in the sugar-binding pocket. The structure is the best determined alpha-D-glucoside:Con A complex to date and the hydrogen bonding network in the saccharide-binding site can be described with some confidence and compared with that of the alpha-D-mannosides.

Conformational change of the adenovirus DNA-binding protein induced by soaking crystals with K3UO2F5 solutions.

Soaking crystals of the C-terminal DNA-binding domain of the adenovirus single-stranded DNA-binding protein with a buffer containing K(3)UO(2)F(5) results in a 9% change of the crystallographic c axis without destruction of the crystals or appreciable loss of resolution. The crystals belong to space group P2(1)2(1)2(1) with a = 79.7, b = 75.6 and c = 60.6 A. The three-dimensional structure has been refined to 2.7 A with a crystallographic R factor of 0.206. Antiparallel chains of protein molecules running through the entire crystal are linked by uranyl ions. The relative orientation of protein monomers is flexible, even in the crystalline state, and allows changes in the packing of the protein chains.

The crystal structure of the complexes of concanavalin A with 4'-nitrophenyl-alpha-D-mannopyranoside and 4'-nitrophenyl-alpha-D-glucopyranoside.

Concanavalin A (Con A) is the best-known plant lectin and has important in vitro biological activities arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complexes of Con A with 4'-nitrophenyl-alpha-D-mannopyranoside (alpha-PNM) and 4'-nitrophenyl-alpha-D-glucopyranoside (alpha-PNG) have been crystallized in space group P2(1)2(1)2 with cell dimensions a = 135.19 A, b = 155.38 A, c = 71.25 A and a = 134.66 A, b = 155.67 A, and c = 71.42 A, respectively. X-ray diffraction intensities to 2.75 A for the alpha-PNM and to 3.0 A resolution for the alpha-PNG complex have been collected. The structures of the complexes were solved by molecular replacement and refined by simulated annealing methods to crystallographic R-factor values of 0.185/0.186 and free-R-factor values of 0.260/0.274, respectively. In both structures, the asymmetric unit contains four molecules arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site near the surface of each monomer. The nonsugar (aglycon) portion of the compounds used helps to identify the exact orientation of the saccharide in the sugar-binding pocket and is involved in major interactions between tetramers. The hydrogen bonding network in the region of the binding site has been analyzed, and only minor differences with the previously reported Con A-methyl-alpha-D-mannopyranoside complex structure have been observed. Structural differences that may contribute to the slight preference of the lectin for mannosides over glucosides are discussed. Calculations indicate a negative electrostatic surface potential for the saccharide binding site of Con A, which may be important for its biological activity. It is also shown in detail how a particular class of hydrophobic ligands interact with one of the three so-called characteristic hydrophobic sites of the lectins.

Alternative arrangements of the protein chain are possible for the adenovirus single-stranded DNA binding protein.

A second crystal form of the C-terminal domain of the adenovirus single-stranded DNA binding protein crystallizes in space group P2(1)2(1)2(1) with a=61.0 angstrom, b=91.2 angstrom and c=149.4 angstrom. The crystals contain two molecules per asymmetric unit and diffract to a maximum resolution of 3.0 angstrom. The crystal is composed of infinite chains of molecules along the crystallographic 2(1) axis parallel to c. The principal intermolecular interaction is a hooking of the C-terminal 17 residues of one molecule onto the next molecule in the protein chain. Adjacent molecules in the chain are rotated approximately 90 degrees with respect to their neighbours. The difference in relative orientation of adjacent molecules between the two crystal forms of the protein implies a degree of flexibility in the protein chain that would facilitate DNA binding.

Crystallization and preliminary X-ray crystallographic studies on the adenovirus ssDNA binding protein in complex with ssDNA.

Crystals of the C-terminal domain of the adenovirus single-stranded DNA binding protein (DBP) in complex with the single-stranded oligonucleotide (dT)16 have been obtained by a batch method from material obtained by chymotryptic digest of full-length DBP. The colorless crystals grow as hexagonal prisms to a maximal size of approximately 0.85 x 0.55 x 0.55 mm. The spacegroup is P3(1)21 with unit cell constants a = 151.5 A and c = 124.0 A. There are either three or four molecules of DBP per asymmetrical unit. To improve the reproducibility of crystallization, recombinant protein has been produced using a baculovirus expression system and shown to crystallize under the same conditions.