Michel Laguerre

Intramolecular and Intermolecular Interactions of Protein Kinase B Define Its Activation In Vivo

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention.

Iterative design of a helically folded aromatic oligoamide sequence for the selective encapsulation of fructose

The ab initio design of synthetic molecular receptors for a specific biomolecular guest remains an elusive objective, particularly for targets such as monosaccharides, which have very close structural analogues. Here we report a powerful approach to produce receptors with very high selectivity for specific monosaccharides and, as a demonstration, we develop a foldamer that selectively encapsulates fructose. The approach uses an iterative design process that exploits the modular structure of folded synthetic oligomer sequences in conjunction with molecular modelling and structural characterization to inform subsequent refinements. Starting from a first-principles design taking size, shape and hydrogen-bonding ability into account and using the high predictability of aromatic oligoamide foldamer conformations and their propensity to crystallize, a sequence that binds to β-D-fructopyranose in organic solvents with atomic-scale complementarity was obtained in just a few iterative modifications. This scheme, which mimics the adaptable construction of biopolymers from a limited number of monomer units, provides a general protocol for the development of selective receptors.

Molecular Structure of Self-Assembled Chiral Nanoribbons and Nanotubules Revealed in the Hydrated State

A detailed molecular organization of racemic 16-2-16 tartrate self-assembled multi-bilayer ribbons in the hydrated state is proposed where 16-2-16 amphiphiles, tartrate ions, and water molecules are all accurately positioned by comparing experimental X-ray powder diffraction and diffraction patterns derived from modeling studies. X-ray diffuse scattering studies show that molecular organization is not fundamentally altered when comparing the flat ribbons of the racemate to chirally twisted or helical ribbons of the pure tartrate enantiomer. Essential features of the three-dimensional molecular organizations of these structures include interdigitation of alkyl chains within each bilayer and well-defined networks of ionic and hydrogen bonds between cations, anions, and water molecules between bilayers. The detailed study of diffraction patterns also indicated that the gemini headgroups are oriented parallel to the long edge of the ribbons. The structure thus possesses a high cohesion and good crystallinity, and for the first time, we could relate the packing of the chiral molecules to the expression of the chirality at a mesoscopic scale. The organization of the ribbons at the molecular level sheds light on a number of their macroscopic features. Among these are the reason why enantiomerically pure 16-2-16 tartrate forms ribbons that consist of exactly two bilayers, and a plausible mechanism by which a chirally twisted or helical shape may emerge from the packing of chiral tartrate ions. Importantly, the distinction between commonly observed helical and twisted morphologies could be related to a subtle symmetry breaking. These results demonstrate that accurately solving the molecular structure of self-assembled soft materials--a process rarely achieved--is within reach, that it is a valid approach to correlate molecular parameters to macroscopic properties, and thus that it offers opportunities to modulate properties through molecular design.

NMR and molecular modeling of wine tannins binding to saliva proteins: revisiting astringency from molecular and colloidal prospects

In organoleptic science, the association of tannins to saliva proteins leads to the poorly understood phenomenon of astringency. To decipher this interaction at molecular and colloidal levels, the binding of 4 procyanidin dimers (B1–4) and 1 trimer (C2) to a human saliva proline‐rich peptide, IB714, was studied. Interactions have been characterized by measuring dissociation constants, sizes of complexes, number, and nature of binding sites using NMR (chemical shift variations, diffusion‐ordered spectroscopy, and saturation transfer diffusion). The binding sites were identified using molecular mechanics, and the hydrophilic/hydrophobic nature of the interactions was resolved by calculating the molecular lipophilicity potential within the complexes. The following comprehensive scheme can be proposed: 1) below the tannin critical micelle concentration (CMC), interaction is specific, and the procyanidin anchorage always occurs on the same three IB714 sites. The tannin 3‐dimensional structure plays a key role in the binding force and in the tannins ability to act as a bidentate ligand: tannins adopting an extended conformation exhibit higher affinity toward protein and initiate the formation of a network. 2) Above the CMC, after the first specific hydrophilic interaction has taken place, a random hydrophobic stacking occurs between tannins and proteins. The whole process is discussed in the general frame of wine tannins eliciting astringency.—Cala, O., Pinaud, N., Simon, C., Fouquet, E., Laguerre, M., Dufourc, E. J., Pianet, I. NMR and molecular modeling of wine tannins binding to saliva proteins: revisiting astringency from molecular and colloidal prospects. FASEB J. 24, 4281–4290 (2010). www.fasebj.org

Silylation d'hydrocarbures mono-aromatiques mono- ou disubstitues

Abstract In the presence of lithium and in tetrahydrofuran as solvent, trimethylchlorosilane reacts at 0–10°C, in an inert atmosphere, with monoaromatic mono- or disubstituted hydrocarbons, to give the corresponding 1,4-disilyl derivatives. In the case of ortho -xylene and cumene, disilylation is accompanied by tetrasilylation, whereas phenyltrimethylsilane can lead to the tetrasilylated derivative in quantitative yields. 1,3-Dichlorotetramethyldisiloxane reacts with benzene to give: Most of the products described are new compounds.

Mechanisms of tannin-induced trypsin inhibition: a molecular approach.

Association of procyanidins with enzymes has drawn attention over the past few years. This work aimed to bring insights on interaction of the protease trypsin with the procyanidin dimer (B3). This interaction was characterized by fluorescence quenching, saturation transfer difference (STD) NMR, molecular modeling, and through an enzymatic inhibition assay. Further studies were conducted regarding the influence of pectin on the binding process. A general overview of the binding process may be outlined as follows: a) at low procyanidin concentrations (below the critical micellar concentration-(CMC)) a specific interaction probably driven by hydrogen bonds between the protein backbone and the procyanidin occurs and is associated with the reduction of both enzyme activity and fluorescence; b) at high procyanidin concentration (above the CMC) the interaction becomes nonspecific. This variation in both nature and extent of the interaction with the variation of procyanidin concentration shows how tannin self-association may affect the interaction between tannins and proteins. It was also shown that the mechanism through which pectin affects the interaction between procyanidin B3 and trypsin is of a competitive type.

Modeling Procyanidin Self-Association Processes and Understanding Their Micellar Organization : A Study by Diffusion NMR and Molecular Mechanics

The colloidal behavior of eight synthetic procyanidins (three monomers, four dimers, and a trimer) has been investigated in water or in a winelike medium using DOSY NMR spectroscopy and molecular dynamics simulations. Different behavior was observed for monomers and oligomers. Monomers self-associate with a high affinity constant (37-53 M(-1)) to form micelles at low cmc (critical micelle concentration) values (1-5 g.L(-1)). These micelles undergo a time-dependent coalescence process to form hazes and precipitates. As for dimers and the trimer, self-association also occurs but with a lower affinity (approximately 6 M(-1)) and at higher cmc values (10-20 g.L(-1)) to form small micelles (<5 nm) that remain stable throughout the experiment. The presence of 10% ethanol does not significantly affect the self-association constant for monomers and oligomers but increases their cmc values by approximately 50% and decreases the micelle size by a factor 2. However, the presence of 20 mM NaCl appears to negate the effect of ethanol. This study helps to clarify the role of procyanidin monomers versus oligomers in wine turbidity and demonstrates that procyanidin oligomers are fully available to interact with saliva proteins.

Long-range effects on the capture and release of a chiral guest by a helical molecular capsule.

Helically folded molecular capsules based on oligoamide sequences of aromatic amino acids which are capable of binding tartaric acid in organic solvents with high affinity and diastereoselectivity have been synthesized, and their structures and binding properties investigated by (1)H NMR, X-ray crystallography, circular dichroism, and molecular modeling. We found that elongating the helices at their extremities by adding monomers remote from the tartaric binding site results in a strong increase of the overall helix stability, but it does not influence the host-guest complex stability. The effect of this elongation on the binding and release rates of the guest molecules follows an unexpected non-monotonous trend. Three independent observations (direct monitoring of exchange over time, 2D-EXSY NMR, and molecular modeling) concur and show that guest exchange rates tend to first increase upon increasing helix length and then decrease when helix length is increased further. This investigation thus reveals the complex effects of adding monomers in a helically folded sequence on a binding event that occurs at a remote site and sheds light on possible binding and release mechanisms.

Structure of a Complex Formed by a Protein and a Helical Aromatic Oligoamide Foldamer at 2.1 Å Resolution

In the search of molecules that could recognize sizeable areas of protein surfaces, a series of ten helical aromatic oligoamide foldamers was synthesized on solid phase. The foldamers comprise three to five monomers carrying various proteinogenic side chains, and exist as racemic mixtures of interconverting right-handed and left-handed helices. Functionalization of the foldamers by a nanomolar ligand of human carbonic anhydrase II (HCA) ensured that they would be held in close proximity to the protein surface. Foldamer-protein interactions were screened by circular dichroism (CD). One foldamer displayed intense CD bands indicating that a preferred helix handedness is induced upon interacting with the protein surface. The crystal structure of the complex between this foldamer and HCA could be resolved at 2.1 Å resolution and revealed a number of unanticipated protein-foldamer, foldamer-foldamer, and protein-protein interactions.

3-D structure and dynamics of protein kinase B—new mechanism for the allosteric regulation of an AGC kinase

New developments regarding the structure and in vivo dynamics of protein kinase B (PKB/Akt) have been recently exposed. Here, we specifically review how the use of multi-disciplinary approaches has resulted in reaching the recent progress made to relate the quaternary structure of PKB to its in vivo function. Using X-ray crystallography, the structure of PKB pleckstrin homology (PH) and kinase domains was determined separately. The molecular mechanisms involved in (a) the binding of the phosphoinositides to the PH domain and (b) the activation of the kinase with the rearrangement of the catalytic site and substrate binding were determined. In vitro, nuclear magnetic resonance and circular dychroism studies gave complementary information on the interaction of the PH domain with the phosphoinositides. However, the molecular nature and the function of the interactions between the PKB domains could not be deduced from the X-ray data since the full-length PKB has not been crystallised. In vitro, dynamic information on the inter-domain conformational changes related to PKB activation states emerged with the use of tandem mass spectrometry. Cell imaging and Förster resonance energy transfer provided in vivo dynamics. Molecular modelling and dynamic simulations in conjunction with mutagenesis and biochemical analysis were used to investigate the complex interactions between the PKB domains in vivo and understand at the molecular level how it linked to its activity. The compilation of the information obtained on the 3-D structure and the spatiotemporal dynamics of this widely studied oncogene could be applied to the study of other proteins. This inter-disciplinary approach led to a more profound understanding of PKB complex activation mechanism in vivo that will shed light onto new ideas and possibilities for modulating its activity.