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Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 Å from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions. [ABSTRACT FROM AUTHOR]

The oncoprotein E7 of human papilloma viruses (HPV) is involved in the pathogenesis and maintenance of human cervical cancers. The most prevalent HPV types found in cervix carcinomas are HPV16, 18 and 45. The structure of the E7 dimer from HPV45 (PDB 2F8B) was determined by nuclear magnetic resonance spectroscopy. Each monomer comprises an unfolded N-terminus and a well-structured C-terminal domain with a β1β2α1β3α2 topology representing a unique zinc-binding fold found only for E7. Dimerization occurs through the α1/α1′ helices and intermolecular β-sheet formation but excludes the zinc-binding sites. E7 is reported to interact with a number of cellular proteins (e.g. pRb, p21CIP1). Binding of a peptide derived from the C-terminus of p21CIP1 to the C-terminal domain of E7 was characterized by monitoring chemical shift perturbations of the amide groups of E7. This provides direct evidence that a shallow groove situated between α1 and β1 of the E7 C-terminal domain is interacting with the C-terminus of p21CIP1. Intriguingly, this binding site overlaps with the low-affinity binding site on E7 for the C-domain of pRb.Oncogene (2006) 25, 5953–5959. doi:10.1038/sj.onc.1209584; published online 24 April 2006 [ABSTRACT FROM AUTHOR]

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c7, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c7, which is paramagnetic with three paramagnetic centers, has been determined via 1H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 ± 0.17 Å and 1.05 ± 0.14 Å for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short β-sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 ± 0.07 Å and 0.92 ± 0.06 Å for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c7 with [Fe] hydrogenase, inducing a change in Km. This finding is in agreement with the identification of the protein area around heme IV as the interacting site. [ABSTRACT FROM AUTHOR]

As protein crystals generally possess a high water content, it is assumed that the behaviour of a protein in solution and in crystal environment is very similar. This assumption can be investigated by molecular dynamics (MD) simulation of proteins in the different environments. Two 2ns simulations of hen egg white lysozyme (HEWL) in crystal and solution environment are compared to one another and to experimental data derived from both X-ray and NMR experiments, such as crystallographic B-factors, NOE atom–atom distance bounds, 3JH Nα-coupling constants, and 1H-15N bond vector order parameters. Both MD simulations give very similar results. The crystal simulation reproduces X-ray and NMR data slightly better than the solution simulation. [ABSTRACT FROM AUTHOR]

The three-dimensional structure in solution of the reduced form of cytochrome c from the green alga Monoraphidium braunii has been solved through NMR data. Cytochrome c acts as a small mono-heme electron carrier protein between the two membrane-embedded complexes cytochrome f and photosystem I. The structure was determined using 1278 relevant interproton NOEs out of 1776 assigned NOEs with distance geometry (DG) calculations which included 36 stereospecific assignments and 20 experimentally found angle constraints. The family of structures obtained from the DG calculations was subjected to energy minimization and molecular dynamics simulation using previously defined force field parameters for the heme and its ligands. In all stages of the calculations, the solution structure is well defined and similar to the now available X-ray structure. [ABSTRACT FROM AUTHOR]

A synthetic peptide, the N-terminus of hepatitis B virus surface antigen Pre-S1, was studied by twodimensional NMR techniques. A series of1H nuclear magnetic resonance experiments were used to complete the identification of spin systems and sequential assignments of this 28-residue peptide. 157 distance constraints and 55 dihedral angle constraints were obtained. 20 structures with the lowest target function were selected by the distance geometry program DIANA. Energy minimization and the following 100 ps time-averaged restrained molecular dynamics (TR-MD) simulation in aqueous solution were performed for each conformer. After TRMD simulation, three locally convergent regions corresponding to residues 22–31, 36–40, 41–46 were found. The averaged pairwise root-meansquare deviation (RMSD) of backbone atoms for them were (1.71±0.49)A, (0.76 ±0.31)A, ( 1.05 ± 0.52)A, respectively. Four reverse turns found in these regions, residues 22–25, 37–40, 41–44 and 43–46, correspond to several important antibody binding sites revealed in relevant immunological research. [ABSTRACT FROM AUTHOR]