Jose Antonio Garate

Conformationally Constrained Lipid A Mimetics for Exploration of Structural Basis of TLR4/MD-2 Activation by Lipopolysaccharide

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on β,α-trehalose-type scaffold. Replacement of the flexible three-bond β(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond β,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.

Computational modeling study of functional microdomains in cannabinoid receptor type 1

The seven transmembrane helices (TMH) G-protein-coupled receptors (GPCRs) constitute one of the largest superfamily of signaling proteins found in mammals. Some of its members, in which the cannabinoid (CB) receptors are included, stand out because their functional states can be modulated by a broad spectrum of effector molecules. The relative ligand promiscuity exhibited by these receptors could be related with particular attributes conferred by their molecular architecture and represents a motivating issue to be explored. In this regard, this study represents an effort to investigate the cannabinoid receptor type 1 (CB1) ligand recognition plasticity, using comparative modeling, molecular dynamics (MD) simulations and docking. Our results suggest that a cooperative set of subtle structural rearrangements within the TMHs provide to the CB1 protein the plasticity to reach alternate configurations. These changes include the relaxation of intramolecular constraints, the rotations, translations and kinks of the majority of TMHs and the reorganization of the ligand binding cavities.

Lipid A from lipopolysaccharide recognition: structure, dynamics and cooperativity by molecular dynamics simulations.

Molecular dynamics simulations of Lipid A and its natural precursor Lipid IVA from E.coli have been carried out free in solution, bound to the myeliod differentiation protein 2 (MD2) and in the complex of MD2 with the toll like receptor 4 (TLR4). In addition, simulations of the ligand free MD2 and MD2‐TLR4 complex were performed. A structural and energetic characterization of the bound and unbound states of Lipid A/IVA was generated. As the crystal structures depict, the main driving force for MD2‐Lipid A/IVA are the hydrophobic interactions between the aliphatic tails and the MD2 cavity. The charged phosphate groups do strongly interact with positively charged residues, located at the surface of MD2. However, they are not essential for keeping the lipids in the cavity, indicating a more prominent role in binding recognition and ionic interactions with TLR4 at the MD2/TLR4 interface. Interestingly, in the absence of any ligand MD2 rapidly closes, blocking the binding cavity. The presence of TLR4, though changing the dynamics, was not able to impede the aforementioned closing event. We hypothesize that fluctuations of the H1 region are essential for this phenomenon, and it is plausible that an equilibrium between the open and closed states exists, although the lengths of our simulations are not sufficient to encompass the reversible process. The MD2/Lipid A‐TLR4 complex simulations show that the presence of the ligand energetically stabilizes the complex relative to the ligand‐free structures, indicating cooperativity in the binding process.

Free‐energy differences between states with different conformational ensembles

Multiple conformations separated by high‐energy barriers represent a challenging problem in free‐energy calculations due to the difficulties in achieving adequate sampling. We present an application of thermodynamic integration (TI) in conjunction with the local elevation umbrella sampling (LE/US) method to improve convergence in alchemical free‐energy calculations. TI‐LE/US was applied to the guanosine triphosphate (GTP) to 8‐Br‐GTP perturbation, molecules that present high‐energy barriers between the anti and syn states and that have inverted preferences for those states. The convergence and reliability of TI‐LE/US was assessed by comparing with previous results using the enhanced‐sampling one‐step perturbation (OSP) method. A linear interpolation of the end‐state biasing potentials was sufficient to dramatically improve sampling along the chosen reaction coordinate. Conformational free‐energy differences were also computed for the syn and anti states and compared to experimental and theoretical results. Additionally, a coupled OSP with LE/US was carried out, allowing the calculation of conformational and alchemical free energies of GTP and 8‐substituted GTP analogs.

Entropic and Enthalpic Contributions to Stereospecific Ligand Binding from Enhanced Sampling Methods

The stereoselective binding of R- and S-propranolol to the metabolic enzyme cytochrome P450 2D6 and its mutant F483A was studied using various computational approaches. Previously reported free-energy differences from Hamiltonian replica exchange simulations, combined with thermodynamic integration, are compared to the one-step perturbation approach, combined with local-elevation enhanced sampling, and an excellent agreement between methods was obtained. Further, the free-energy differences are interpreted in terms of enthalpic and entropic contributions where it is shown that exactly compensating contributions obscure a molecular interpretation of differences in the affinity while various reduced terms allow a more detailed analysis, which agree with heuristic observations on the interactions.

Anti-endotoxic activity and structural basis for human MD-2·TLR4 antagonism of tetraacylated lipid A mimetics based on βGlcN(1↔1)αGlcN scaffold

Interfering with LPS binding by the co-receptor protein myeloid differentiation factor 2 (MD-2) represents a useful approach for down-regulation of MD-2·TLR4-mediated innate immune signaling, which is implicated in the pathogenesis of a variety of human diseases, including sepsis syndrome. The antagonistic activity of a series of novel synthetic tetraacylated bis-phosphorylated glycolipids based on the βGlcN(1↔1)αGlcN scaffold was assessed in human monocytic macrophage-like cell line THP-1, dendritic cells and human epithelial cells. Two compounds were shown to inhibit efficiently the LPS-induced inflammatory signaling by down-regulation of the expression of TNF-α, IL-6, IL-8, IL-10 and IL-12 to background levels. The binding of the tetraacylated by (R)-3-hydroxy-fatty acids (2 × C12, 2 × C14), 4,4′-bisphosphorylated βGlcN(1↔1)αGlcN-based lipid A mimetic DA193 to human MD-2 was calculated to be 20-fold stronger than that of Escherichia coli lipid A. Potent antagonistic activity was related to a specific molecular shape induced by the β,α(1↔1)-diglucosamine backbone. ‘Co-planar’ relative arrangement of the GlcN rings was inflicted by the double exo-anomeric conformation around both glycosidic torsions in the rigid β,α(1↔1) linkage, which was ascertained using NOESY NMR experiments and confirmed by molecular dynamics simulation. In contrast to the native lipid A ligands, the binding affinity of βGlcN(1↔1)αGlcN-based lipid A mimetics to human MD-2 was independent on the orientation of the diglucosamine backbone of the synthetic antagonist within the binding pocket of hMD-2 (rotation by 180°) allowing for two equally efficient binding modes as shown by molecular dynamics simulation.

From dimers to collective dipoles: Structure and dynamics of methanol/ethanol partition by narrow carbon nanotubes

Alcohol partitioning by narrow single-walled carbon nanotubes (SWCNTs) holds the promise for the development of novel nanodevices for diverse applications. Consequently, in this work, the partition of small alcohols by narrow tubes was kinetically and structurally quantified via molecular dynamics simulations. Alcohol partitioning is a fast process in the order of 10 ns for diluted solutions but the axial-diffusivity within SWCNT is greatly diminished being two to three orders of magnitude lower with respect to bulk conditions. Structurally, alcohols form a single-file conformation under confinement and more interestingly, they exhibit a pore-width dependent transition from dipole dimers to a single collective dipole, for both methanol and ethanol. Energetic analyses demonstrate that this transition is the result of a detailed balance between dispersion and electrostatics interactions, with the latter being more pronounced for collective dipoles. This transition fully modifies the reorientational dynamics of the loaded particles, generating stable collective dipoles that could find usage in signal-amplification devices. Overall, the results herein have shown distinct physico-chemical features of confined alcohols and are a further step towards the understanding and development of novel nanofluidics within SWCNTs.

Exploring the membrane potential of a simple dual membrane system by using a constant electric field

Background Connexins (Cxs) constitute Gap Junction Channels (GJCs). GJCs connect the cytoplasm of adjacent cells providing a hydrophilic path between cells that allow the movement, by passive diffusion, of water, cations and small molecules. The opening or closing of GJCs is dependent on the voltage difference between the apposed cells and/or the membrane potential. An approach to understand the voltage gating mechanisms of GJCs is to study a simplified system that can account for the basic features of a GJC.