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.

Atomistic mechanism of leptin and leptin-receptor association.

The leptin-leptin receptor complex is at the very core of energy homeostasis and immune system regulation, among many other functions. In this work, we built homology models of leptin and the leptin binding domain (LBD) of the receptor from humans and mice. Docking analyses were used to obtain the coordinates of the native leptin-LBD complexes and a mixed heterodimer formed by human leptin and mouse LBD. Molecular dynamics (MD) simulations were performed using all models (monomers and heterodimers) as initial coordinates and the GROMACS program. The overall structural and dynamical behaviors are similar for the three complexes. Upon MD simulations, several new interactions appear. In particular, hydrophobic interactions, with more than 90% persistence, seem to be the most relevant for the stability of the dimers, as well as the pair formed by Asp85Lep and Arg468LBD. This in silico analysis provides structural and dynamical information, at the atomistic level, about the mechanism of leptin-LBD complex formation and leptin receptor activation. This knowledge might be used in the rational drug design of therapeutics to modulate leptin signaling.Communicated by Ramaswamy H. Sarma.

Artificial Intelligence Algorithms in Visual Evoked Potential-Based Brain-Computer Interfaces for Motor Rehabilitation Applications: Systematic Review and Future Directions.

Brain-Computer Interface (BCI) is a technology that uses electroencephalographic (EEG) signals to control external devices, such as Functional Electrical Stimulation (FES). Visual BCI paradigms based on P300 and Steady State Visually Evoked potentials (SSVEP) have shown high potential for clinical purposes. Numerous studies have been published on P300- and SSVEP-based non-invasive BCIs, but many of them present two shortcomings: (1) they are not aimed for motor rehabilitation applications, and (2) they do not report in detail the artificial intelligence (AI) methods used for classification, or their performance metrics. To address this gap, in this paper the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) methodology was applied to prepare a systematic literature review (SLR). Papers older than 10 years, repeated or not related to a motor rehabilitation application, were excluded. Of all the studies, 51.02% referred to theoretical analysis of classification algorithms. Of the remaining, 28.48% were for spelling, 12.73% for diverse applications (control of wheelchair or home appliances), and only 7.77% were focused on motor rehabilitation. After the inclusion and exclusion criteria were applied and quality screening was performed, 34 articles were selected. Of them, 26.47% used the P300 and 55.8% the SSVEP signal. Five applications categories were established: Rehabilitation Systems (17.64%), Virtual Reality environments (23.52%), FES (17.64%), Orthosis (29.41%), and Prosthesis (11.76%). Of all the works, only four performed tests with patients. The most reported machine learning (ML) algorithms used for classification were linear discriminant analysis (LDA) (48.64%) and support vector machine (16.21%), while only one study used a deep learning algorithm: a Convolutional Neural Network (CNN). The reported accuracy ranged from 38.02 to 100%, and the Information Transfer Rate from 1.55 to 49.25 bits per minute. While LDA is still the most used AI algorithm, CNN has shown promising results, but due to their high technical implementation requirements, many researchers do not justify its implementation as worthwile. To achieve quick and accurate online BCIs for motor rehabilitation applications, future works on SSVEP-, P300-based and hybrid BCIs should focus on optimizing the visual stimulation module and the training stage of ML and DL algorithms.

Transit of Procaspase-9 towards its activation. New mechanistic insights from molecular dynamics simulations.

Caspases are cysteine proteases that perform a wide variety of roles in lethal intracellular signaling and cell-death regulation. Caspase-9, the primary initiator caspase of the intrinsic apoptotic pathway, is produced as a scarcely active zymogen (Procaspase-9). Here, we describe, for the first time, at the atomistic level, conformational changes which might be correlated to the activation of Procaspase-9. Molecular dynamics simulations performed at two temperatures (310 and 410 K) provide insights about the conformational space and the time-course evolution of the geometrical and structural characteristics of Procaspase-9. At both temperatures studied, the extremal globular domains of the protein approach each other, contracting the disordered region. In both temperatures, the compact conformations hide more than 40 nm2 (about 20% of the total solvent-accessible surface area), and their radius of gyration are reduced by about 40% from the original values. At each temperature, the pathway of contraction is different, as well as the compact structures reached. In consequence, the network of stabilizing interactions at the final conformations is dissimilar. Both final conformations were evaluated in their structural compatibility with the activation models described so far. In this work, we describe mechanistically how and why the activation of Procaspase-9 is favored by apoptosome recruitment via the Caspase Activation Recruitment Domain (CARD), as it has been proposed recently by in vitro experiments.

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.

Studying the collective motions of the adenosine A2A receptor as a result of ligand binding using principal component analysis.

Adenosine receptors (ARs) belong to family A of GPCRs that are involved in many diseases, including cerebral and cardiac ischemic diseases, immune and inflammatory disorders, etc. Thus, they represent important therapeutic targets to treat these conditions. Computational techniques such as molecular dynamics (MD) simulations permit researchers to obtain structural information about these proteins, and principal component analysis (PCA) allows for the identification of collective motions. There are available structures for the active form (3QAK) and the inactive form (3EML) of A2AR which permit us to gain insight about their activation/inactivation mechanism. In this work, we have proposed an inverse strategy using MD simulations where the active form was coupled to the antagonist caffeine and the inactive form was coupled to adenosine agonist. Moreover, we have included four reported thermostabilizing mutations in the inactive form to study A2AR structural differences under different conditions. Some observations stand out from the PCA studies. For instance, the apo structures showed remarkable similarities, and the principal components (PCs) were rearranged in a ligand-dependent manner. Additionally, the active conformation was less stable compared to the inactive one. Some PCs inverted their direction in the presence of a ligand, and comparison of the PCs between 3EML and 3EML_ADN showed that adenosine induced major changes in the structure of A2AR. Rearrangement of PCs precedes and drives conformational changes that occur after ligand binding. Knowledge about these conformational changes provides important insights about the activity of A2AR.

The importance of evolutionarily conserved C-terminal basic residues for the stability of proapoptotic Bax protein.

Bax is a protein that promotes apoptosis (a form of cell death). The atomistic details of the mechanism by which Bax is activated during apoptosis remain a subject of debate. C-terminal basic residues in the sequence of Bax show remarkable conservation across a variety of species. The role of these charged residues in the stability of Bax was investigated by submitting substituted mutants to molecular dynamics simulations at high temperatures. Mutation of either or both K189 and K190 led to dramatic changes in helical content, radius of gyration, proximity of the C terminus to the core of the protein, exposure of the BH3 domain, and bundling of the core. These results suggest a critical role of positively charged residues close to the C terminus in the structural stability of Bax.

Mapping the intrinsically disordered properties of the flexible loop domain of Bcl-2: a molecular dynamics simulation study.

Most of the B-cell lymphoma-2 (Bcl-2) protein structure has been elucidated; however, the conformation of its flexible loop domain (FLD) has not yet been experimentally predicted. Its high flexibility under physiological conditions is the reason. FLD behaves as an intrinsically disordered region (IDR) and can adopt regular structures in particular conditions associated with the control of Bcl-2's anti-apoptotic functions. In a previous contribution, we analyzed an engineered Bcl-2 construct (Bcl-2-Δ22Σ3) submitted to 25-ns MD and reported a disordered-to-helix transitions in a region of FLD (rFLD, residues 60-77). However, the conformational preferences in solution of rFLD in the nanosecond to microsecond scale were not analyzed. Herein, an average model was obtained for the native Bcl-2 protein by homology modeling and MD simulation techniques. From this, only the atomic coordinates corresponding to the rFLD were simulated for 1 µs by MD at 310 K. In concordance with previous studies, a disordered-to-helix transitions were exhibited, implying that this "interconversion of folding" in the rFLD suggest a possible set of conformations encoded in its sequence. Principal component analysis (PCA) showed that most of the conformational fluctuation of Bcl-2 is provided by rFLD. Dihedral PCA (dPCA) offered information about all the conformations of rFLD in the µs of the simulation, characterizing a dPCA-based free energy landscape of rFLD, and a conformational ensemble of fast interconverting conformations as other IDRs. Furthermore, despite the conformational heterogeneity of rFLD, the analysis of the dihedral angles (Φ, Ψ) showed that this region does not randomly explore the conformational space in solution.

Exploring the structure and conformational landscape of human leptin. A molecular dynamics approach.

Leptin is a hormone that regulates energy homeostasis, inflammation, hematopoiesis and immune response, among other functions (Houseknecht et al., 1998; Zhang et al., 1995; Paz-Filho et al., 2010). To obtain its crystallographic structure, it was necessary to substitute a tryptophan for a glutamic acid at position 100, thus creating a mutant leptin that has been reported to have biological activity comparable to the activity of the wild type but that crystallizes more readily. Here, we report a comparative study of the conformational space of WT and W100E leptin using molecular dynamics simulations performed at 300, 400, and 500 K. We detected differences between the interactions of the two proteins with local and distal effects, resulting in changes in the conformation, accessible surface area, compactness, electrostatic potential and dynamic behavior. Additionally, the series of unfolding events that occur when leptin is subjected to high temperature differs for the two constructs. We observed that both proteins are mostly unstructured after 20 ns of MD simulation at 500 K. However, WT leptin maintains a significant amount of secondary structure in helix α2, while the most stable region of W100E leptin is helix α3. Furthermore, we found that the region between residues 25 and 42 might adopt interconverting secondary structures ranging from α-helices and random coils to ß-strand structures. Thus, this region can be considered an intrinsically disordered region. This atomistic description supports our understanding of leptin signaling and consequently might facilitate the use of leptin in treatments for the pathophysiologies in which it is implicated.

A continuous peptide epitope reacting with pandemic influenza AH1N1 predicted by bioinformatic approaches.

Computational identification of potential epitopes with an immunogenic capacity challenges immunological research. Several methods show considerable success, and together with experimental studies, the efficiency of the algorithms to identify potential peptides with biological activity has improved. Herein, an epitope was designed by combining bioinformatics, docking, and molecular dynamics simulations. The hemagglutinin protein of the H1N1 influenza pandemic strain served as a template, owing to the interest of obtaining a scheme of immunization. Afterward, we performed enzyme-linked immunosorbent assay (ELISA) using the epitope to analyze if any antibodies in human sera before and after the influenza outbreak in 2009 recognize this peptide. Also, a plaque reduction neutralization test induced by virus-neutralizing antibodies and the IgG determination showed the biological activity of this computationally designed peptide. The results of the ELISAs demonstrated that the serum of both prepandemic and pandemic recognized the epitope. Moreover, the plaque reduction neutralization test evidenced the capacity of the designed peptide to neutralize influenza virus in Madin-Darby canine cells.