In silico EsxG EsxH rational epitope selection: Candidate epitopes for vaccine design against pulmonary tuberculosis.

Rational design of new vaccines against pulmonary tuberculosis is imperative. Early secreted antigens (Esx) G and H are involved in metal uptake, drug resistance, and immune response evasion. These characteristics make it an ideal target for rational vaccine development. The aim of this study is to show the rational design of epitope-based peptide vaccines by using bioinformatics and structural vaccinology tools. A total of 4.15 µs of Molecular Dynamics simulations were carried out to describe the behavior in solution of heterodimer, single epitopes, and epitopes loaded into MHC-II complexes. In order to predict T and B cell epitopes for antigenic activation, bioinformatic tools were used. Hence, we propose three epitopes with the potential to design pulmonary tuberculosis vaccines. The possible use of the proposed epitopes includes subunit vaccines, as a booster in BCG vaccination to improve its immune response, as well as the generation of antibodies that interfere with the Mycobacterium tuberculosis homeostasis, affecting its survival.

Anti-apoptotic Bcl-2 protein in apo and holo conformation anchored to the membrane: comparative molecular dynamics simulations.

The interaction between the anti-apoptotic Bcl-2 protein and its antagonist Bax is essential to the regulation of the mitochondrial pathway of apoptosis. For this work, we built models by homology of Bcl-2 full-sequence length in monomeric form (apo-Bcl-2) and in complex with the BH3 domain of Bax (holo-Bcl-2). The Bcl-2 protein was analyzed with its transmembrane domain anchored to a lipidic bilayer of DPPC, imitating physiological conditions. We performed molecular dynamics (MD) simulations using the GROMACS program. Conformational changes showed that the flexible loop domain (FLD) tends to fold on itself and move towards the main core. Furthermore, the BH3 peptide of pro-apoptotic protein Bax, showed an allosteric stabilizing effect on FLD upon being bound to the hydrophobic cleft of the anti-apoptotic protein Bcl-2, causing a reduction in its structural flexibility. However, FLD is distal from the main core of Bcl-2. Principal component analysis (PCA) showed a weak correlation between FLD residues and BH3 peptide from Bax. Upon MD simulations, several new contacts appeared between FLD and some α-helices of the core of Bcl-2, which contribute to maintaining the stability of Bcl-2. This knowledge sheds light on the behavior of Bcl-2 in the cell's native environment.Communicated by Ramaswamy H. Sarma.

Exploring the Conformational Space of Bcl-2 Protein Variants: Dynamic Contributions of the Flexible Loop Domain and Transmembrane Region.

Members of the Bcl-2 protein family regulate apoptosis through interactions with several proteins. A critical intrinsically disordered region (IDR) present in some members of the Bcl-2 family is essential for their function. Also, the structural and conformational plasticity of disordered regions is essential for the regulation of the Bcl-2 protein's activity. Further, some proteins of the family contain transmembrane-helical regions, which anchor them into organelle membranes. Bcl-2, the archetypical member of the family, is characterized by an IDR labeled as a flexible loop domain (FLD) and a transmembrane domain (TMD). Another member of this family is the Bcl-2A1 protein, containing a TMD but lacking the FLD. To our knowledge, this is the first report which characterizes the individual and simultaneous dynamical contributions of FLD and TMD in Bcl-2 and Bcl-2A1 using molecular dynamics simulations (MDS). We examined the conformational spaces of Bcl-2, Bcl-2A1, and two artificial constructs lacking the TMD (Bcl-2ΔTM and Bcl-2A1ΔTM). As the results show, FLD and TMD stabilized each protein independently when they are present. When they coincided, such as in Bcl-2, an additive stabilizing effect is observed. This information is crucial for understanding the structural mechanisms of interaction in the Bcl-2 family.

Chemical and Immunological Characteristics of Aluminum-Based, Oil-Water Emulsion, and Bacterial-Origin Adjuvants.

Adjuvants are a diverse family of substances whose main objective is to increase the strength, quality, and duration of the immune response caused by vaccines. The most commonly used adjuvants are aluminum-based, oil-water emulsion, and bacterial-origin adjuvants. In this paper, we will discuss how the election of adjuvants is important for the adjuvant-mediated induction of immunity for different types of vaccines. Aluminum-based adjuvants are the most commonly used, the safest, and have the best efficacy, due to the triggering of a strong humoral response, albeit generating a weak induction of cell-mediated immune response. Freund's adjuvant is the most widely used oil-water emulsion adjuvant in animal trials; it stimulates inflammation and causes aggregation and precipitation of soluble protein antigens that facilitate the uptake by antigen-presenting cells (APCs). Adjuvants of bacterial origin, such as flagellin, E. coli membranes, and monophosphoryl lipid A (MLA), are known to potentiate immune responses, but their safety and risks are the main concern of their clinical use. This minireview summarizes the mechanisms that classic and novel adjuvants produce to stimulate immune responses.

Cyclo-lib: a database of computational molecular dynamics simulations of cyclodextrins.

MOTIVATION: Cyclodextrins (CDs) are amongst the most versatile/multi-functional molecules used in molecular research and chemical applications. They are natural cyclic oligosaccharides typically employed to encapsulate hydrophobic groups in their central cavity. This allows solubilizing, protecting or reducing the toxicity of a large variety of different molecules including drugs, dyes and surfactant agents. In spite of their great potential, atomic level information of these molecules, which is key for their function, is really scarce. Computational Molecular Dynamics (MD) simulations have the potential to efficiently fill this gap, providing structural-dynamic information at atomic level in time scales ranging from ps to µs. RESULTS: Cyclo-lib is a database with a publicly accessible web-interface containing structural and dynamic analysis obtained from computational MD simulation trajectories (250 ns long) of native and modified CDs in explicit water molecules. Cyclo-lib currently includes 70 CDs typically employed for fundamental and industrial research. Tools for comparative analysis between different CDs, as well as to restrict the analysis to specific time-segments within the trajectories are also available. Cyclo-lib provides atomic resolution information aimed to complement experimental results performed with the same molecules. AVAILABILITY AND IMPLEMENTATION: The database is freely available under http://cyclo-lib.mduse.com/ CONTACT: Angel.Pineiro@usc.es.

Surface adsorption and bulk aggregation of cyclodextrins by computational molecular dynamics simulations as a function of temperature: α-CD vs ß-CD.

The structural simplicity of native cyclodextrins (CDs) contrasts with their complex behavior in the bulk of aqueous solutions, mainly when they are combined with other cosolutes. Many scientific and industrial applications based on these molecules are supported only by empirical information. The lack of fundamental knowledge, which would allow one to rationally optimize many of these applications, is notable mainly at the solution/air interface. Basic information on phenomena such as the spontaneous adsorption of native CDs or on the structure of CD aggregates in the bulk solution is really scarce. In order to fill these gaps, a detailed computational study on the adsorption and aggregation of α- and ß-CDs as a function of temperature is presented here. Our simulations reproduce, at atomic resolution, the experimentally observed much higher ability of ß-CD to aggregate compared to that of α-CD at 298 K, as well as their dependence on temperature. The adsorption of both individual CDs and small CD aggregates (up to 20 molecules) to the solution/air interface is found to be negligible. 0.8 µs long trajectories of single CD molecules in aqueous solution reveal that the main differences in the behavior of both CDs are their flexibility, higher for ß-CD, and the occupancy of individual intramolecular hydrogen bonds that is significantly longer for the same cyclodextrin. The aggregation pattern of α- and ß-CDs is followed at the hundreds of ns time scale, allowing both the spontaneous self-assembly of cyclodextrins and their redistribution along the aggregates to be observed. This is the first attempt to study the adsorption and aggregation of native cyclodextrins by atomistic molecular dynamics simulations.

Theoretical analysis of the catalytic mechanism of Helicobacter pylori glutamate racemase.

One of the most challenging open key questions behind the stereoinversion of D-glutamate and L-glutamate catalyzed by glutamate racemases is how those enzymes manage to generate the thermodynamically unfavorable reverse protonation state of the catalytic residue cysteine required for the proton abstraction from the α-carbon of glutamate. In this paper, we have used molecular dynamics (MD) simulations with a molecular mechanics force field along with QM/MM calculations starting from the crystal structure and from different MD snapshots to study the enantiomeric conversion of D-glutamate to L-glutamate catalyzed by the Helicobacter pylori glutamate racemase. Our results show that structural fluctuations of the enzyme-substrate complex, represented by the different snapshots, lead to reaction paths with different features and fates. The whole reaction, when it occurs, involves four successive proton transfers in three or four different steps. In the first step, Asp7 assists the deprotonation of D-glutamate by participating in general base catalysis with neutral Cys70 thiol. An analogous mechanism was previously found by some of us for the case of Bacillus subtilis glutamate racemase. This fact explains why that aspartate belongs to the group of strictly conserved residues.

How the substrate D-glutamate drives the catalytic action of Bacillus subtilis glutamate racemase.

Molecular Dynamics simulations with a Molecular Mechanics force field and a quite complete exploration of the QM/MM potential energy surfaces have been performed to study the D-glutamate --> L-glutamate reaction catalyzed by Bacillus subtilis glutamate racemase. The results show that the whole process involves four successive proton transfers that occur in three different steps. The Michaelis complex is already prepared to make the first proton transfer (from Cys74 to Asp10) possible. The second step involves two proton transfers (from the alpha-carbon to Cys74, and from Cys185 to the alpha-carbon), which occurs in a concerted way, although highly asynchronic. Finally, in the third step, the nascent deprotonated Cys185 is protonated by His187. The positively charged ammonium group of the substrate plays a very important key role in the reaction. It accompanies each proton transfer in a concerted and coupled way, but moving itself in the opposite direction from Asp10 to His187. Thus, the catalytic action of Bacillus subtilis glutamate racemase is driven by its own substrate of the reaction, D-glutamate.