Serge Crouzy

Gating of retinal rod cation channel by different nucleotides : comparative study of unitary currents

SummarySingle channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K0.5 is about 3 μm for 8Br-cGMP, 18 μm for cGMP and 740 μm for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni2+ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMPor 8Br-cGMP dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry28:2785–2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases Po; addition of cAMP concentrations higher than about 70 μm progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol.123:133–147).

The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA

The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the “open” conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Å were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.

A Gadolinium-Binding Cyclodecapeptide with a Large High-Field Relaxivity Involving Second-Sphere Water

A new cyclodecapeptide incorporating two prolylglycine sequences as beta-turn inducers and bearing four side chains with acidic carboxyl groups for cation complexation has been prepared. Structural analysis in water by (1)H NMR spectroscopy and CD shows that this template adopts a conformation suitable for the complexation of lanthanide ions Ln(3+), with its carboxyl groups oriented on the same face of the peptide scaffold. Luminescence titrations show that mononuclear Ln-PA complexes are formed with apparent stability constants of log beta(110) approximately 6.5 (pH 7). The high-field water relaxivity values arising from the Gd-PA complex at 200-500 MHz have been interpreted with molecular parameters determined independently. The experimentally determined water relaxivities are undoubtedly 30% higher than the expected values for this complex with two inner-sphere (IS) water molecules and a medium-range rotational correlation time (tau(R) = 386 ps (+/-10%)). This led us to propose the existence of a large second-sphere (2S) contribution to the relaxivity caused by the interaction of water molecules with the hydrophilic peptide ligand by hydrogen-bonding.

Outer-Sphere Investigation of MRI Relaxation Contrast Agents. Example of a Cyclodecapeptide Gadolinium Complex with Second-Sphere Water

We show how the purely outer-sphere (OS) relaxivity of a probe solute due to a Gd(3+) complex can help characterize the outer (O), inner (I), and second (2) sphere (S) contributions to the water proton relaxivity. Because of the difficulties of accurate theoretical predictions, we propose an experimental determination of the OS dipolar time correlation function (OS-DTCF) of the relative position of Gd(3+) with respect to any of the equivalent protons of the purely OS probe p-dioxane, which moves around the complex without binding to it. The method is illustrated by the GdPA complex with PA = c(AspArgGluProGlyGluTrpAspProGly). The experimental DTCF for dioxane is obtained by a model-free analysis of the high-field relaxivity of its protons. The time-modulation of the dioxane DTCF by the Gd(3+) electronic spin relaxation yields a measurable quenching of the longitudinal relaxivity at low-to-medium field, which serves us to deduce the fluctuating zero-field splitting (ZFS) Hamiltonian causing this electronic relaxation. The DTCF for water is derived from that for dioxane by appropriate scaling of the geometry of collision and relative diffusion coefficients of these molecules with respect to GdPA. The information obtained on the OS motion for water and the ZFS Hamiltonian together with an independent characterization of the IS contribution allows us to disentangle the OS, IS, and 2S mechanisms and interpret the relaxivity profile of the water protons from 2.35 mT to 18.8 T. The presence of a large 2S contribution is confirmed.

4-Hydroxyphenylpyruvate Dioxygenase Catalysis IDENTIFICATION OF CATALYTIC RESIDUES AND PRODUCTION OF A HYDROXYLATED INTERMEDIATE SHARED WITH A STRUCTURALLY UNRELATED ENZYME

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

Adaptive torsion-angle quasi-statics

MOTIVATION The cost of molecular quasi-statics or dynamics simulations increases with the size of the simulated systems, which is a problem when studying biological phenomena that involve large molecules over long time scales. To address this problem, one has often to either increase the processing power (which might be expensive), or make arbitrary simplifications to the system (which might bias the study). RESULTS We introduce adaptive torsion-angle quasi-statics, a general simulation method able to rigorously and automatically predict the most mobile regions in a simulated system, under user-defined precision or time constraints. By predicting and simulating only these most important regions, the adaptive method provides the user with complete control on the balance between precision and computational cost, without requiring him or her to perform a priori, arbitrary simplifications. We build on our previous research on adaptive articulated-body simulation and show how, by taking advantage of the partial rigidification of a molecule, we are able to propose novel data structures and algorithms for adaptive update of molecular forces and energies. This results in a globally adaptive molecular quasi-statics simulation method. We demonstrate our approach on several examples and show how adaptive quasi-statics allows a user to interactively design, modify and study potentially complex protein structures.

Simulation Analysis of the Retinal Conformational Equilibrium in Dark-Adapted Bacteriorhodopsin

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect.

Interplay between glutathione, Atx1 and copper: X-ray absorption spectroscopy determination of Cu(I) environment in an Atx1 dimer.

X-ray absorption techniques have been used to characterise the primary coordination sphere of Cu(I) bound to glutathionate (GS−), to Atx1 and in Cu2I(GS−)2(Atx1)2, a complex recently proposed as the major form of Atx1 in the cytosol. In each complex, Cu(I) was shown to be triply coordinated. When only glutathione is provided, each Cu(I) is triply coordinated by sulphur atoms in the binuclear complex CuI2(GS−)5, involving bridging and terminal thiolates. In the presence of Atx1 and excess of glutathione, under conditions where CuI2(GS−)2(Atx1)2 is formed, each Cu(I) is triply coordinated by sulphur atoms. Given these constraints, there are two different ways for Cu(I) to bridge the Atx1 dimer: either both Cu(I) ions contribute to bridging the dimer, or only one Cu(I) ion is responsible for bridging, the other one being coordinated to two glutathione molecules. These two models are discussed as regards Cu(I) transfer to Ccc2a.

Copper(II)-Complex Directed Regioselective Mono-p-Toluenesulfonylation of Cyclomaltoheptaose at a Primary Hydroxyl Group Position: An NMR and Molecular Dynamics-Aided Design

Interactions between cyclomaltoheptaose (β-cyclodextrin, βCD) and p-toluenesulfonyl chloride (TsCl) were investigated using MD simulations, both in vacuum, approximating the hydrophobic environment of the CD cavity, and with water as a solvent. In both cases, the minimum energy adiabatic paths, and the mean force potentials (MFP) for the insertion of TsCl along a reaction coordinate perpendicular to the CD plane, were calculated for the two possible orientations of TsCl. The results show a preferred entry of TsCl in the CD cavity with the sulfonyl chloride group pointing to the primary hydroxyls rim. In each orientation, two energy minima for the complex are detected in vacuum that reflect the H-H contacts between host and guest observed by NMR spectroscopy (ROESY, NOESY). These separate minima collapsed into a single broader minimum, when the solvent was introduced in the simulations. The resulting association constant between TsCl and βCD (K(a) ≈ 100 M(-1)) is in good agreement with the NMR results (K(a) = 102 ± 12 M(-1)) in deuterated water solution at 298 K. Advantage has been taken of the dynamics of the reagent inclusion to set up a one step process involving a transient Cu(2+) chelate at the secondary hydroxyls rim position for the electrophilic monoactivation of βCD at the primary hydroxyls rim using water as solvent.

Molecular dynamics simulations of the ErbB-2 transmembrane domain within an explicit membrane environment: comparison with vacuum simulations

Two 500-ps molecular dynamics simulations performed on the single transmembrane domain of the ErbB-2 tyrosine kinase receptor immersed in a fully solvated dilauroylphosphatidyl-ethanolamine bilayer (DLPE) are compared to vacuum simulations. One membrane simulation shows that the initial alpha helix undergoes a local pi helix conversion in the peptide part embedded in the membrane core similar to that found in simulation vacuum. Lipid/water/peptide interaction analysis shows that in the helix core, the intramolecular peptide interactions are largely dominant compared to the interactions with water and lipids whereas the helix extremities are much more sensitive to these interactions at the membrane interfaces. Our results suggest that simulations in a lipid environment are required to understand the dynamics of transmembrane helices, but can be reasonably supplemented by in vacuo simulations to explore rapidly its conformational space and to describe the internal deformation of the hydrophobic core.