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Antibody affinity maturation is one of the fundamental processes of immune defense against invading pathogens. From the biological point of view, the clonal selection hypothesis represents the most accepted mechanism to explain how mutations increasing the affinity for target antigens are introduced and selected in antibody molecules. However, understanding at the molecular level how protein modifications, such as point mutation, can modify and modulate the affinity of an antibody for its antigen is still a major open issue in molecular biology. In this paper, we address various aspects of this problem by analyzing and comparing atomistic simulations of 17 variants of the bevacizumab antibody, all directed against the common target protein VEGF-A. In particular, we examine MD-based descriptors of the internal energetics and dynamics of mutated antibodies and their possible correlations with experimentally determined affinities for the antigens. Our results show that affinity improvement is correlated with a variation of the internal stabilization energy of the antibody molecule when bound to the antigen, compensated by the variation in the interaction energy between the antigen and the antibody, paralleled by an overall modulation of internal coordination within the antibody molecular structure. A possible model of the mechanism of rigidification and of the main residues involved is proposed. Overall, our results can help in understanding the molecular determinants of antigen recognition and have implications in the rational design of new antibodies with optimized affinities.

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR., Author Summary Computational modeling combined with experimental validation may give insight into structural and functional properties of protein systems. The basic Helix-Loop-Helix PER-ARNT-SIM (bHLH-PAS) proteins show conserved functional domains despite the broad range of functions exerted by the different systems. Within this protein family, the Aryl hydrocarbon Receptor (AhR) is known to mediate the toxic effects of a number of environmental contaminants, including dioxins and dioxin-like chemicals, and it also exerts other biochemical and physiological effects. Despite the absence of experimentally determined structures, theoretical models of the AhR PAS domains developed on the basis of homologous systems have allowed understanding of some aspects of the molecular mechanisms underlying its function. In this work we present alternative structural models of the transcriptionally active complex of AhR with the AhR Nuclear Translocator (ARNT) protein. Computational characterization of the modeled protein-protein interaction interfaces guided the design of mutagenesis experiments, and evaluation of the DNA binding ability of the resulting AhR:ARNT dimer mutants allowed validation of the models and selection of the most reliable one. These findings open new research directions for understanding the molecular mechanisms underlying the functional activity of the AhR.

Unraveling the molecular basis of immune recognition still represents a challenging task for current biological sciences, both in terms of theoretical knowledge and practical implications. Here, we describe the physical-chemistry methods and computational protocols for the prediction of antibody-binding epitopes and MHC-II loaded epitopes, starting from the atomic coordinates of antigenic proteins (PDB file). These concepts are the base of the Web tool BEPPE (Binding Epitope Prediction from Protein Energetics), a free service that returns a list of putative epitope sequences and related blast searches against the Uniprot human complete proteome. BEPPE can be employed for the study of the biophysical processes at the basis of the immune recognition, as well as for immunological purposes such as the rational design of biomarkers and targets for diagnostics, therapeutics, and vaccine discovery.

MicroRNAs (miRNAs) are potent modulators of protein expression that play key roles in brain pathways regulating neurogenesis and synaptic plasticity. These small RNAs may be critical for the pathophysiology of mental disorders and may influence the effectiveness of psychotropic drugs. To investigate the possible involvement of miRNAs in the mechanism of action of antidepressants (AD), we conducted a whole-miRNome quantitative analysis with qRT-PCR of the changes in the blood of 10 depressed subjects after 12 weeks of treatment with escitalopram. Thirty miRNAs were differentially expressed after the AD treatment: 28 miRNAs were up-regulated, and 2 miRNAs were strongly down-regulated. miRNA target gene prediction and functional annotation analysis showed that there was a significant enrichment in several pathways associated with neuronal brain function (such as neuroactive ligand-receptor interaction, axon guidance, long-term potentiation and depression), supporting the hypothesis that the differentially regulated miRNAs may be involved in the AD mechanism. (C) 2012 Elsevier B.V. and ECNP. All rights reserved.