Order parameters of a transmembrane helix in a fluid bilayer: case study of a WALP peptide.

A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including (13)C and (15)N chemical shift anisotropies and (13)C-(15)N dipolar couplings, were determined from two different triple-isotope-labeled WALP23 peptides ((2)H, (13)C, and (15)N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21 degrees was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5 degrees previously determined from (2)H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error.

Structural insights on the pamoic acid and the 8 kDa domain of DNA polymerase beta complex: towards the design of higher-affinity inhibitors.

BACKGROUND: DNA polymerase beta (pol beta), the error-prone DNA polymerase of single-stranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis. For this reason pol beta has become a therapeutic target. Several inhibitors have been identified, but none of them presents a sufficient affinity and specificity to become a drug. The fragment-based inhibitor design allows an important improvement in affinity of small molecules. The initial and critical step for setting up the fragment-based strategy consists in the identification and structural characterization of the first fragment bound to the target. RESULTS: We have performed docking studies of pamoic acid, a 9 micromolar pol beta inhibitor, and found that it binds in a single pocket at the surface of the 8 kDa domain of pol beta. However, docking studies provided five possible conformations for pamoic acid in this site. NMR experiments were performed on the complex to select a single conformation among the five retained. Chemical Shift Mapping data confirmed pamoic acid binding site found by docking while NOESY and saturation transfer experiments provided distances between pairs of protons from the pamoic acid and those of the 8 kDa domain that allowed the identification of the correct conformation. CONCLUSION: Combining NMR experiments on the complex with docking results allowed us to build a three-dimensional structural model. This model serves as the starting point for further structural studies aimed at improving the affinity of pamoic acid for binding to DNA polymerase beta.

Structure-function analysis of the THAP zinc finger of THAP1, a large C2CH DNA-binding module linked to Rb/E2F pathways.

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of approximately 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel beta-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.

Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments.

Order parameters from deuterium NMR are often used to validate or calibrate molecular dynamics simulations. This paper gives a short overview of the literature in which experimental order parameters from (2)H NMR are compared to those calculated from MD simulations. The different ways in which order parameters from experiment are used to calibrate and validate simulations are reviewed. In the second part of this review, a case study of cholesterol in a DMPC bilayer is presented. It is concluded that the agreement between experimental data and simulation is favorable in the hydrophobic region of the membrane, for both the phospholipids and cholesterol. In the interfacial region the agreement is less satisfactory, probably because of the high polarity of this region which makes the correct computation of the electrostatics more complex.

Upregulation of error-prone DNA polymerases beta and kappa slows down fork progression without activating the replication checkpoint.

There is rising evidence that cancer development is associated from its earliest stages with DNA replication stress, a major source of spontaneous genomic instability. However, the origin of these replication defects has remained unclear. We have investigated the consequences of upregulating error-prone DNA polymerases (pol) beta and kappa on chromosomal DNA replication. These enzymes are misregulated in different types of cancers and induce major chromosomal instabilities when overexpressed at low levels. Here, we have used DNA combing to show that a moderate overexpression of pol beta or pol kappa is sufficient to impede replication fork progression and to promote the activation of additional replication origins. Interestingly, alterations of the normal replication program induced by excess error-prone polymerases were not detected by the replication checkpoint. We therefore propose that upregulation of error-prone DNA polymerases induces a checkpoint-blind replication stress that contributes to genomic instability and to cancer development.

Differential binding to the alpha/beta-tubulin dimer of vinorelbine and vinflunine revealed by nuclear magnetic resonance analyses.

The binding of two antitumour alkaloids, vinorelbine and vinflunine, to the alpha/beta-tubulin dimer has been investigated at equilibrium by nuclear magnetic resonance (NMR) spectroscopy. Tubulin stability and assembly induced by these drugs has been checked under NMR experimental conditions, and tubulin spirals were found in majority. Then, using increasing ligand concentrations, the alkaloids were titrated against tubulin. A non-specific binding of both compounds to tubulin (K(d)>10(-5)M) was characterised by broad NMR ligand signal at 4 and 30 degrees. The tubulin dimer exhibited also 2.7 (sigma: 0.3) and 2.6 (sigma: 0.6) binding sites with a K(d)<10(-5)M for vinorelbine at 4 and 30 degrees, respectively. In contrast, if the tubulin dimer exhibited 2.7 (sigma: 0.2) binding sites for vinflunine at 4 degrees, these sites were not detected at 30 degrees. This NMR study revealed for the first time the presence of specific binding sites and a clear differential affinity of vinorelbine and vinflunine to the tubulin dimer at physiological temperatures which could possibly account for their differential cytotoxicity.