A model of the complex between single-stranded DNA and the single-stranded DNA binding protein encoded by gene V of filamentous bacteriophage M13.

A contact analysis and a series of restrained molecular dynamics simulations were employed to derive a model of the complex between single-stranded DNA and the single-stranded DNA-binding protein encoded by gene V of the filamentous phage M13. The study is based on the recently elucidated solution structure of the Tyr41-->His mutant of the protein. Electron microscopy studies, indicating that the complex forms a flexible, left-handed helical coil with a diameter of 8 to 9 nm and an average pitch of 9 nm, were taken into consideration. The contact analysis served to determine the helix parameters that permit the energetically most favourable packing of protein molecules. Then a protein super-helix was built, into which two extended strands of DNA were modelled using restrained molecular dynamics. Specific constraints were included to ensure that the DNA would position itself into the binding groove of the protein. These constraints are based on recent NMR spin label experiments which offered a direct identification of the amino acids of the protein present in the DNA-binding domain. We present a model for the complex which is in full agreement with the existing reliable biophysical and biochemical data. A description of the protein-protein interface is given and the protein-DNA interaction is discussed in view of the derived model. In addition, we demonstrate that, on the basis of the available experimental data, and not imposing the left-handedness of the nucleoprotein complex, it is feasible to build also a plausible model for the complex which exhibits the opposite, i.e. right-handed, helical sense. This nucleoprotein structure features characteristics highly similar to those of the left-handed helix.

The solution structure of the Tyr41-->His mutant of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13.

The solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been investigated by nuclear magnetic resonance spectroscopy. Two- and three-dimensional NMR experiments have been employed with a variety of NMR samples of gene V protein, some of which were uniformly enriched with either 15N or 13C. The structure of mutant Tyr41-->His of the M13 gene V protein which occurs in solution as a symmetric dimer was calculated using a two-stage procedure. The first step of the procedure involved the calculation of a set of individual monomer structures using the distance geometry program DIANA. This was then followed by the calculation of dimer structures employing "simulated annealing" protocols with the program X-PLOR. Hereby, the problem of assignment of intra- and inter-subunit NOEs of the symmetric dimer was circumvented through use of a target function that correctly deals with the intra- and inter-subunit contributions to the NOE peaks. Furthermore, a pseudo energy term was employed to restrain the symmetry of the dimer. In addition to this novel calculation strategy, we have incorporated distance information for a set of NOEs which were unambiguously identified as inter-subunit NOEs using an NMR strategy based on asymmetric labelling. A total of 20 structures were calculated for the M13 gene V protein mutant Tyr41-->His based on approximately 1000 experimental restraints derived from the NMR data. The structure of residues 1 to 15 and 29 to 87 of both monomers is reasonably well determined with an average atomic r.m.s. difference between the individual structures and the respective mean structure of approximately 0.9 A for the backbone atoms and approximately 1.4 A for all atoms. The orientation of the exposed anti-parallel beta-loop (residues 16 to 28) with respect to the core could not be determined. The molecular architecture of each of the monomers includes a five-stranded beta-barrel enclosing a hydrophobic core and two-antiparallel beta-loops. The dimer structure is stabilized predominantly by hydrophobic residues primarily involving the symmetry-related dyad domains (residues 64 to 82) of the monomers. Residues which are close to bound single-stranded DNA were identified previously from binding experiments with spin-labelled oligonucleotides. The solution structure of mutant Tyr41-->His of the M13 gene V protein is consistent with these binding data and provides a clear view of the protein's single-stranded DNA binding path.

Solution structure of the M13 major coat protein in detergent micelles: a basis for a model of phage assembly involving specific residues.

The three-dimensional structure of the major coat protein of bacteriophage M13, solubilized in detergent micelles, has been determined using heteronuclear multidimensional NMR and restrained molecular dynamics. The protein consists of two alpha-helices, running from residues 8 to 16 and 25 to 45, respectively. These two helices are connected by a flexible and distorted helical hinge region. The structural properties of the coat protein make it resemble a flail, in which the hydrophobic helix (residues 25 to 45) is the handle and the other, amphipathic, helix the swingle. In this metaphor, the hinge region is the connecting piece of leather. The mobility of the residues in the hinge region is likely to enable a smooth transformation from the membrane-bound form, mimicked by the structure in detergent micelles, into the structure in the mature phage. A specific distribution of the residues over the surface of the two helices was observed in the presented high-resolution structure of the membrane-bound form of the major coat protein as well as in the structure in the mature phage. All data suggest that this arrangement of residues is important for the interactions of the protein with the membrane, for correct protein-DNA and protein-protein interactions in the phage and for a proper growth of the phage during the assembly process. By combining our findings with earlier NMR results on the major coat protein in detergent micelles, we were able to construct a model that addresses the role of specific residues in the assembly process.

Single-stranded DNA binding protein encoded by the filamentous bacteriophage M13: structural and functional characteristics.

The single-stranded DNA binding protein, or gene V protein (gVp), encoded by gene V of the filamentous bacteriophage M13 is a multifunctional protein that not only regulates viral DNA replication but also gene expression at the level of mRNA translation. It furthermore is implicated as a scaffolding and/or chaperone protein during the phage assembly process at the hostcell membrane. The protein is 87 amino acids long and its biological functional entity is a homodimer. In this manuscript a short description of the life cycle of filamentous phages is presented and our current knowledge of the major functional and structural properties and characteristics of gene V protein are reviewed. In addition models of the superhelical complexes gVp forms with ssDNA are described and their (possible) biological meaning in the infection process are discussed. Finally it is described that the 'DNA binding loop' of gVp is a recurring motif in many ssDNA binding proteins and that the fold of gVp is shared by a large family of evolutionarily conserved gene regulatory proteins.

Floating stereospecific assignment revisited: application to an 18 kDa protein and comparison with J-coupling data.

We report a floating chirality procedure to treat nonstereospecifically assigned methylene or isopropyl groups in the calculation of protein structures from NMR data using restrained molecular dynamics and simulated annealing. The protocol makes use of two strategies to induce the proper conformation of the prochiral centres: explicit atom 'swapping' following an evaluation of the NOE energy term, and atom 'floating' by reducing the angle and improper force constants that enforce a defined chirality at the prochiral centre. The individual contributions of both approaches have been investigated. In addition, the effects of accuracy and precision of the interproton distance restraints were studied. The model system employed is the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophage Pf3. Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-coupling data. We conclude that floating stereospecific assignment is a reliable tool in protein structure calculation. Its use is beneficial because it allows the distance restraints to be extracted directly from the measured peak volumes without the need for averaging or adding pseudoatom corrections. As a result, the calculated structures are of a quality almost comparable to that obtained with stereospecific assignments. As floating chirality furthermore is the only approach treating prochiral centres that ensures a consistent assignment of the two proton frequencies in a single structure, it seems to be preferable over using pseudoatoms or (R(-6)) averaging.

A (13)C double-filtered NOESY with strongly reduced artefacts and improved sensitivity.

A (1)H NOESY experiment with two (13)C half-filters is described which has, compared to previously reported versions, an enhanced overall sensitivity and strongly reduced intramolecular cross peaks in any part of the spectrum edited for intermolecular NOEs. By adding a shaped (13)C pulse to the half-filter which selectively inverts the aromatic resonances, the filter can be tuned separately and simultaneously for the aliphatic and aromatic regions. Contrary to recently proposed schemes, no magnetization is destroyed, so that full sensitivity is retained for symmetric systems such as homodimers. Furthermore, by replacing the rectangular 180° (13)C pulses by high-power hyperbolic secant pulses for inversion of the complete (13)C spectral range, offset effects (which are another source of signal loss and artefacts) are eliminated. The spectra edited for intermolecular NOEs clearly demonstrate that residual artefacts are considerably smaller than in the original version of the experiment.

Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.

The solution structure of the synthetic circular peptide CGVSRQGKPYC. NMR studies of the folding of a synthetic model for the DNA-binding loop of the ssDNA-binding protein encoded by gene V of phage M13.

The cyclic disulfide peptide CGVSRQGKPYC was prepared to obtain a constrained analogue of residues 17-27 of the DNA-binding loop of the gene-V-encoded sDNA-binding protein of filamentous bacteriophage M13. Amino acid sequences very similar to that of the beta-loop have been found in various phage-encoded ssDNA-binding proteins, and it has been proposed that such a loop may occur as a common motif in this class of proteins. The conformation, in aqueous solution, of the synthetic gene-V-protein binding-loop analogue has been investigated by means of two-dimensional-1H-NMR techniques. Subsequent structure calculations show that the molecule forms a beta-loop that includes a turn formed by three residues. This structure, very unusually for a cyclic disulfide peptide, is highly similar to that of the analogous part of the binding loop of the native protein. Comparison with experiments on other cyclic disulfide peptides indicates that the formation, of the beta-sheet (beta-hairpin) secondary structure is essentially governed by the amino acid composition of the 11-residue sequence. The disulfide bridge in the 11-residue sequence is essential for conformational stability, as indicated by the finding that the open peptide analogue that encompasses residues Ser17-Ser27 does not adopt a detectable secondary structure in water. The bridge replaces the role of the loop formed by residues 49-58 in the protein, which act as a scaffold to hold the N-terminal and C-terminal ends of the DNA-binding loop together.

Refined structure, DNA binding studies, and dynamics of the bacteriophage Pf3 encoded single-stranded DNA binding protein.

The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-edited NOESY spectra and many homo- and heteronuclear J-couplings. The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denoted the DNA binding wing with respect to the core of the protein. Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE. It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6. This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed. Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA. Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restrained molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex. Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed that is in accordance with the experimental data.

Refined solution structure of the Tyr41-->His mutant of the M13 gene V protein. A comparison with the crystal structure.

The three-dimensional solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been refined in two stages. The first stage involved the collection of additional NOE-based distance constraints, which were then used in eight cycles of back-calculations and structure calculations. The structures of the gene V protein dimers were calculated using simulated annealing, employing restrained molecular dynamics with a geometric force field. In the second stage of the refinement procedure, solvent was explicitly included during the dynamic calculations. A total of 30 structures was calculated for the protein, representing its solution structure in water. The first calculation step significantly improved the convergence of the structures, whereas the subsequent simulations in water made the structures physically more realistic. This is, for instance, illustrated by the number of hydrogen bonds formed in the molecule, which increased considerably upon going to aqueous solution. It is shown that the solution structure of the mutant gene V protein is nearly identical to the crystal structure of the wild-type molecule, except for the DNA-binding loop (residues 16-28). This antiparallel beta-hairpin is twisted and partially folded back towards the core of the protein in the NMR structure, whereas it is more extended and points away from the rest of the molecule in the X-ray structure. Unrestrained molecular dynamics calculations suggest that this latter conformation is energetically unstable in solution.

Secondary structure of the single-stranded DNA binding protein encoded by filamentous phage Pf3 as determined by NMR.

Nuclear magnetic resonance spectroscopy was employed to study the single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3. The protein is 78 amino acids long and occurs in solution predominantly as a homodimer with a molecular mass of 18 kDa. Sequence-specific 1H and 15N resonance assignments have been obtained using homo- and heteronuclear two- and three-dimensional experiments. The secondary structure of the protein monomer was determined from a qualitative interpretation of nuclear Overhauser enhancement spectra and amide exchange data. It consists of a five-stranded antiparallel beta-sheet and three beta-hairpins. Problems caused by the protein's tendency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36-->His) which exhibits significantly improved solubility characteristics over the wild-type protein. It is shown that this mutation only locally affects the structure of the protein; the chemical shifts of the wild-type and mutant species differ only for a few residues near the site of the mutation, and the secondary structures of the proteins are identical. The secondary structure of the Pf3 single-stranded DNA binding protein is compared to that of the Ff gene V protein, the only single-stranded DNA binding protein for which the complete three-dimensional structure is known to date [Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N. H. & Hilbers, C. W. (1994) J. Mol. Biol. 236, 229-246; Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M., Gray, C. W., Konings, R. N. H., Wang, A. H.-J. & Terwilliger, T. C. (1994) Proc. Natl Acad. Sci. USA 91, 2071-2075]. It is found that the secondary structures of the two proteins are very similar which supports the hypothesis that a five-stranded antiparallel beta-sheet with protruding beta-hairpins is a common motif in a certain class of single-stranded DNA binding proteins. In addition, the sequence and folding predicted earlier for the DNA binding wing in the single-stranded DNA binding protein of phage Pf3 [de Jong, E. A. M., van Duynhoven, J. P. M., Harmsen, B. J. M., Tesser, G. I., Konings, R. N. H. & Hilbers, C. W. (1989) J. Mol. Biol. 206, 133-156] is borne out by the present study. It closely resembles that in the single-stranded DNA binding protein of phage Ff, which may indicate that such a wing is a recurrent motif as well.

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest.

The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 A resolution. We also use NMR spectroscopic titration techniques. This work reveals a novel DNA interaction involving a dimeric 'winged helix' domain protein that differs from predictions. While the two recognition helices of RTP are in close contact with the B-form DNA major grooves, the 'wings' and N-termini of RTP do not form intimate contacts with the DNA. This structure provides insight into the molecular basis of polar replication fork arrest based on a model of cooperative binding and differential binding affinities of RTP to the two adjacent binding sites in the complete terminator.