Alain Milon

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 2H 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.

Comparison of the effects of inserted C40- and C50-terminally dihydroxylated carotenoids on the mechanical properties of various phospholipid vesicles

We have measured the extent of incorporation of zeaxanthin (C40) and decaprenozeaxanthin (C50) in unilamellar vesicles of dimyristoylphosphatidylcholine (n-C14) and dipalmitoylphosphatidylcholine (n-C16). The incorporation is larger when the molecular length of the carotenoid corresponds to the thickness of the phospholipid bilayer. Stereochemically pure 2,3-di-O-phytanyl-sn-glycero-1-phosphocholine was prepared by modification of the polar heads of the phospholipids of Halobacterium halobium. Vesicles of this branched-chain ether phospholipid incorporate poorly the carotenoids, whereas egg lecithin vesicles incorporate them better. Osmotic swelling and water permeability of vesicles, with or without carotenoids, were measured in a stopped-flow, light-scattering system. The reinforcing effect (lower permeability and higher rigidity) of carotenoids at 1.5 mol% incorporation into diphytanylphosphatidylcholine vesicles is comparable to that of 5 mol% cholesterol; however, carotenoids have no measurable effect on the egg lecithin vesicles. These results imply that the reinforcement of the membrane depends on a subtle adjustment of the phospholipid-carotenoid system.

Cholesterol Orientation and Dynamics in Dimyristoylphosphatidylcholine Bilayers: A Solid State Deuterium NMR Analysis

Proton decoupled deuterium NMR spectra of oriented bilayers made of DMPC and 30 mol % deuterated cholesterol acquired at 76.8 MHz (30 degreesC) have provided a set of very accurate quadrupolar splitting for eight C-D bonds of cholesterol. Due to the new precision of the experimental data, the original analysis by. Biochemistry. 23:6062-6071) had to be reconsidered. We performed a systematic study of the influence on the precision and uniqueness of the data-fitting procedure of: (i) the coordinates derived from x-ray, neutron scattering, or force field-minimized structures, (ii) internal mobility, (iii) the axial symmetry hypothesis, and (iv) the knowledge of some quadrupolar splitting assignments. Good agreement between experiment and theory could be obtained only with the neutron scattering structure, for which both axial symmetry hypothesis and full order parameter matrix analysis gave satisfactory results. Finally, this work revealed an average orientation of cholesterol slightly different from those previously published and, most importantly, a molecular order parameter equal to 0.95 +/- 0.01, instead of 0.79 +/- 0.03 previously found for the same system at 30 degreesC. Temperature dependence in the 20-50 degreesC range shows a constant average orientation and a monotonous decrease of cholesterol Smol, with a slope of -0.0016 K-1. A molecular order parameter of 0.89 +/- 0.01 at 30 degreesC was determined for a DMPC/16 mol % of cholesterol.

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 ∼80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel β-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.

Osmotic swelling of unilamellar vesicles by the stopped-flow light scattering method. Influence of vesicle size, solute, temperature, cholesterol and three α,ω-dihydroxycarotenoids

Abstract α,ω-Dihydroxycarotenoids are postulated to play the role of membrane reinforcers in certain procaryotes, like cholesterol in eucaryotes. In order to evaluate the rigidifying effect of these polyterpenoids on lipid bilayers, osmotic swelling of unilamellar vesicles has been followed by measuring the light scattering intensity changes in a stopped-flow apparatus. A model based upon the Rayleigh-Gans theory was developped. It shows that the variation of light scattering intensity ( ΔI I 0 ) is proportional to that of vesicle radius ( ΔR R 0 ) for a given R0 (initial vesicle radius). An emperical relationship between −ΔI I 0 and Z (dissymmetry measured by light scattering) was established: −ΔI I 0 is proportional to (Z − 1). Therefore, the value −ΔI (I 0 · (Z − 1)) is independent of vesicle radius and can be used for the evaluation of bilayer rigidity. The water permeability was measured and it was shown that it is the limiting factor of the kinetics of swelling. When incorporated into dimyristoylphophatidylcholine vesicles, cholesterol and α,ω-dihydroxycarotenoids lower the water permeability and the value of −ΔI (I 0 · (Z − 1)) . So, at least on model systems, α,ω-dihydroxycarotenoids exert on a phospholipid bilayer a reinforcement effect similar to cholesterol.

Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris

The human μ‐opioid receptor cDNA from which the 32 amino‐terminal codons were substituted by the Saccharomyces cerevisiae α‐mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase‐1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (K d = 0.2 nM and B max = 400 fmol/mg protein or 800 sites/cell). Competition studies with non‐selective, and μ‐, δ‐ and κ‐selective opioid agonists and antagonists revealed a typical μ‐opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Green fluorescent protein as a reporter of human μ-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris

Abstract The human μ-opioid receptor (HuMOR) was fused in its N-terminus end to the green fluorescent protein (GFP) or/and to the c-myc and six histidines tags in its C-terminus end, and expressed in the methylotrophic yeast Pichia pastoris. Neither the C- nor the N-terminal tagging of the receptor does modify its pharmacological properties as compared to the untagged receptor. Expression levels of fusion receptors determined by GFP fluorescence measurements strongly correlates with the number of sites expressed per cell detected through saturation studies (Bmax value), thus showing that GFP is an efficient and reliable reporter of the HuMOR functional expression. The N- and C-terminus tags have allowed to show that the entire molecule is overexpressed. They have permitted in-situ localization experiments using fluorescence and electron microscopy techniques and have shown a dense intracellular labelling. Above all, the quantification of expression levels made possible through fluorescence intensity analysis, have revealed that huge amounts of receptor are produced that could not be detected through classical binding experiments: for a Bmax value of 1 pmol mg−1 of receptor determined through binding studies, 16 pmol were found in membrane preparations using fluorescence and 100 pmol in whole cells. These results should be very useful for large-scale production and structural biology of HuMOR, and other G-protein coupled receptors (GPCRs).

Generation of formate by the formyltransferase/hydrolase complex (Fhc) from Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO2. One of the subunits of the complex carries the catalytic site for transfer of the formyl group from tetrahydromethanopterin to methanofuran (MFR). We now found via nuclear magnetic resonance‐based studies that the Ftr complex also catalyzes the hydrolysis of formyl‐MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place.

Structural determinants of specific DNA-recognition by the THAP zinc finger

Human THAP1 is the prototype of a large family of cellular factors sharing an original THAP zinc-finger motif responsible for DNA binding. Human THAP1 regulates endothelial cell proliferation and G1/S cell-cycle progression, through modulation of pRb/E2F cell-cycle target genes including rrm1. Recently, mutations in THAP1 have been found to cause DYT6 primary torsion dystonia, a human neurological disease. We report here the first 3D structure of the complex formed by the DNA-binding domain of THAP1 and its specific DNA target (THABS) found within the rrm1 target gene. The THAP zinc finger uses its double-stranded β-sheet to fill the DNA major groove and provides a unique combination of contacts from the β-sheet, the N-terminal tail and surrounding loops toward the five invariant base pairs of the THABS sequence. Our studies reveal unprecedented insights into the specific DNA recognition mechanisms within this large family of proteins controlling cell proliferation, cell cycle and pluripotency.

Type III Secretion-Dependent Cell Cycle Block Caused in HeLa Cells by Enteropathogenic Escherichia coli O103

ABSTRACT Rabbit enteropathogenic Escherichia coli (EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation. We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms. We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis. A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content. This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation. In contrast, intimin and Tir mutants retained the antiproliferative effect. The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition. Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells. Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1). Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13suc1-agarose beads. This shows that Cdk1 is implicated in the G2/M arrest caused by EPEC strain E22.