Marcelo D. Costabel

Echinococcus granulosus antigen B: a Hydrophobic Ligand Binding Protein at the host-parasite interface.

Lipids are mainly solubilized by various families of lipid binding proteins which participate in their transport between tissues as well as cell compartments. Among these families, Hydrophobic Ligand Binding Proteins (HLBPs) deserve special consideration since they comprise intracellular and extracellular members, are able to bind a variety of fatty acids, retinoids and some sterols, and are present exclusively in cestodes. Since these parasites have lost catabolic and biosynthetic pathways for fatty acids and cholesterol, HLBPs are likely relevant for lipid uptake and transportation between parasite and host cells. Echinococcus granulosus antigen B (EgAgB) is a lipoprotein belonging to the HLBP family, which is very abundant in the larval stage of this parasite. Herein, we review the literature on EgAgB composition, structural organization and biological properties, and propose an integrated scenario in which this parasite HLBP contributes to adaptation to mammalian hosts by meeting both metabolic and immunomodulatory parasite demands.

PKC and PTPα participate in Src activation by 1α,25(OH)2 vitamin D3 in C2C12 skeletal muscle cells

We previously demonstrated that 1α,25(OH)(2)-vitamin D(3) [1α,25(OH)(2)D(3)] induces Src activation, which mediates the hormone-dependent ERK1/2 and p38 MAPK phosphorylation in skeletal muscle cells. In the present study, we have investigated upstream steps whereby 1α,25(OH)(2)D(3) may act to transmit its signal to Src. Preincubation with the PKC inhibitor Ro318220 demonstrated the participation of PKC in 1α,25(OH)(2)D(3)-dependent Src activation. Of interest, the hormone promoted the activation of δ the isoform of PKC. We also explored the role of PTPα in PKC-mediated Src stimulation. Silencing of PTPα with a specific siRNA suppressed Src activation induced by 1α,25(OH)(2)D(3). Hormone treatment increased PTPα (Tyr789) phosphorylation and PKC-dependent phosphatase activity. Accordingly, 1α,25(OH)(2)D(3) promoted serine phosphorylation of PTPα in a PKC-dependent manner. Confocal immunocytochemistry and co-immunoprecipitation assays revealed that the hormone induces the co-localization of Src and PTPα with PKC participation. Computational analysis revealed that the electrostatic interaction between Src and PTPα is favored when PTPα is phosphorylated in Tyr789. These data suggest that 1α,25(OH)(2)D(3) acts in skeletal muscle upstream on MAPK cascades sequentially activating PKC, PTPα and Src.

Prediction of the most favorable configuration in the ACBP-membrane interaction based on electrostatic calculations

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.

IFABP portal region insertion during membrane interaction depends on phospholipid composition.

Intestinal fatty acid-binding protein (IFABP) is highly expressed in the intestinal epithelium and it belongs to the family of soluble lipid binding proteins. These proteins are thought to participate in most aspects of the biology of lipids, regulating its availability for specific metabolic pathways, targeting and vectorial trafficking of lipids to specific subcellular compartments. The present study is based on the ability of IFABP to interact with phospholipid membranes, and we characterized its immersion into the bilayers hydrophobic central region occupied by the acyl-chains. We constructed a series of Trp-mutants of IFABP to selectively probe the interaction of different regions of the protein, particularly the elements forming the portal domain that is proposed to regulate the exit and entry of ligands to/from the binding cavity. We employed several fluorescent techniques based on selective quenching induced by soluble or membrane confined agents. The results indicate that the portal region of IFABP penetrates deeply into the phospholipid bilayer, especially when CL-containing vesicles are employed. The orientation of the protein and the degree of penetration were highly dependent on the lipid composition, the superficial net charge and the ionic strength of the medium. These results may be relevant to understand the mechanism of ligand transfer and the specificity responsible for the unique functions of each member of the FABP family.

A hydrophobic loop in acyl-CoA binding protein is functionally important for binding to palmitoyl-coenzyme A: a molecular dynamics study.

Acyl-CoA binding protein (ACBP) plays a key role in lipid metabolism, interacting via a partly unknown mechanism with high affinity with long chain fatty acyl-CoAs (LCFA-CoAs). At present there is no study of the microscopic way ligand binding is accomplished. We analyzed this process by molecular dynamics (MDs) simulations. We proposed a computational model of ligand, able to reproduce some evidence from nuclear magnetic resonance (NMR) data, quantitative time resolved fluorometry and X-ray crystallography. We found that a hydrophobic loop, not in the active site, is important for function. Besides, multiple sequence alignment shows hydrophobicity (and not the residues itselves) conservation.

Electrostatics of the phospholipase-membrane interaction

The electrostatic interaction of the Phospholipase A2 (PLA2)-membrane complex in the presence and absence of calcium is analysed by the computation of the electrostatic profiles of the components and the complex. The electrostatic potential was computed by using of the program MOLPOT that implement the boundary element method to solve the electrostatic problem. It considers a closed surface in three dimensions that contains the macromolecule that follows as close as possible the macromolecule shape. The results show that the presence of calcium ions contributes to the stability of the complex and at the same time creates a favourable electrostatic potential pattern that may be favourable for the lipolysis of the membrane components.

Multiscale modelization in a small virus: Mechanism of proton channeling and its role in triggering capsid disassembly

In this work, we assess a previously advanced hypothesis that predicts the existence of ion channels in the capsid of small and non-enveloped icosahedral viruses. With this purpose we examine Triatoma Virus (TrV) as a case study. This virus has a stable capsid under highly acidic conditions but disassembles and releases the genome in alkaline environments. Our calculations range from a subtle sub-atomic proton interchange to the dismantling of a large-scale system representing several million of atoms. Our results provide structure-based explanations for the three roles played by the capsid to enable genome release. First, we observe, for the first time, the formation of a hydrophobic gate in the cavity along the five-fold axis of the wild-type virus capsid, which can be disrupted by an ion located in the pore. Second, the channel enables protons to permeate the capsid through a unidirectional Grotthuss-like mechanism, which is the most likely process through which the capsid senses pH. Finally, assuming that the proton leak promotes a charge imbalance in the interior of the capsid, we model an internal pressure that forces shell cracking using coarse-grained simulations. Although qualitatively, this last step could represent the mechanism of capsid opening that allows RNA release. All of our calculations are in agreement with current experimental data obtained using TrV and describe a cascade of events that could explain the destabilization and disassembly of similar icosahedral viruses.

Orthosteric and benzodiazepine cavities of the α1β2γ2 GABAA receptor: insights from experimentally validated in silico methods

γ-aminobutyric acid-type A (GABAA) receptors mediate fast synaptic inhibition in the central nervous system of mammals. They are modulated via several sites by numerous compounds, which include GABA, benzodiazepines, ethanol, neurosteroids and anaesthetics among others. Due to their potential as targets of novel drugs, a detailed knowledge of their structure–function relationships is needed. Here, we present the model of the α1β2γ2 subtype GABAA receptor in the APO state and in complex with selected ligands, including agonists, antagonists and allosteric modulators. The model is based on the crystallographic structure of the human β3 homopentamer GABAA receptor. The complexes were refined using atomistic molecular dynamics simulations. This allowed a broad description of the binding modes and the detection of important interactions in agreement with experimental information. From the best of our knowledge, this is the only model of the α1β2γ2 GABAA receptor that represents altogether the desensitized state of the channel and comprehensively describes the interactions of ligands of the orthosteric and benzodiazepines binding sites in agreement with the available experimental data. Furthermore, it is able to explain small differences regarding the binding of a variety of chemically divergent ligands. Finally, this new model may pave the way for the design of focused experimental studies that will allow a deeper description of the receptor.

Conserved charged amino acids are key determinants for fatty acid binding proteins (FABPs)-membrane interactions. A multi-methodological computational approach

Based on the analysis of the mechanism of ligand transfer to membranes employing in vitro methods, Fatty Acid Binding Protein (FABP) family has been divided in two subgroups: collisional and diffusional FABPs. Although the collisional mechanism has been well characterized employing in vitro methods, the structural features responsible for the difference between collisional and diffusional mechanisms remain uncertain. In this work, we have identified the amino acids putatively responsible for the interaction with membranes of both, collisional and diffusional, subgroups of FABPs. Moreover, we show how specific changes in FABPs’ structure could change the mechanism of interaction with membranes. We have computed protein–membrane interaction energies for members of each subgroup of the family, and performed Molecular Dynamics simulations that have shown different configurations for the initial interaction between FABPs and membranes. In order to generalize our hypothesis, we extended the electrostatic and bioinformatics analysis over FABPs of different mammalian genus. Also, our methodological approach could be used for other systems involving protein–membrane interactions.