Marco D'Abramo

MoDEL (Molecular Dynamics Extended Library): A Database of Atomistic Molecular Dynamics Trajectories

More than 1700 trajectories of proteins representative of monomeric soluble structures in the protein data bank (PDB) have been obtained by means of state-of-the-art atomistic molecular dynamics simulations in near-physiological conditions. The trajectories and analyses are stored in a large data warehouse, which can be queried for dynamic information on proteins, including interactions. Here, we describe the project and the structure and contents of our database, and provide examples of how it can be used to describe the global flexibility properties of proteins. Basic analyses and trajectories stripped of solvent molecules at a reduced resolution level are available from our web server.

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein-DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.

FlexServ: an integrated tool for the analysis of protein flexibility

SUMMARY FlexServ is a web-based tool for the analysis of protein flexibility. The server incorporates powerful protocols for the coarse-grained determination of protein dynamics using different versions of Normal Mode Analysis (NMA), Brownian dynamics (BD) and Discrete Dynamics (DMD). It can also analyze user provided trajectories. The server allows a complete analysis of flexibility using a large variety of metrics, including basic geometrical analysis, B-factors, essential dynamics, stiffness analysis, collectivity measures, Lindemanns indexes, residue correlation, chain-correlations, dynamic domain determination, hinge point detections, etc. Data is presented through a web interface as plain text, 2D and 3D graphics. AVAILABILITY http://mmb.pcb.ub.es/FlexServ; http://www.inab.org

Mixed Quantum-Classical Methods for Molecular Simulations of Biochemical Reactions With Microwave Fields: The Case Study of Myoglobin

Contradictory data in the huge literature on microwaves bio-effects may result from a poor understanding of the mechanisms of interaction between microwaves and biological systems. Molecular simulations of biochemical processes seem to be a promising tool to comprehend microwave induced bio-effects. Molecular simulations of classical and quantum events involved in relevant biochemical processes enable to follow the dynamic evolution of a biochemical reaction in the presence of microwave fields. In this paper, the action of a microwave signal (1 GHz) on the covalent binding process of a ligand (carbon monoxide) to a protein (myoglobin) has been studied. Our results indicate that microwave fields, with intensities much below the atomic/molecular electric interactions, cannot affect such biochemical process.

Conformational Selection versus Induced Fit in Kinases: The Case of PI3K‐γ

Molecular recognition is crucial for a multitude of fundamental biological processes, for example enzyme activation and inhibition, 2] and protein folding. The increasing availability of high-resolution structures of ligands bound to receptors has changed our understanding of molecular recognition from a static concept, in which interactions are considered to be a rigid lock and key, to a dynamic one, in which both the ligand and its target can assume different, yet not always complementary, shapes. Within this dynamic framework, two limiting mechanisms have been proposed: conformational selection 6] and induced fit. The first theory implies that there is a significant overlap between the conformational space that is occupied by the bound and unbound forms of the target, and that the role of the ligand is to stabilize certain conformations that are accessible to the unbound form. In the induced-fit hypothesis, the ligand induces the protein to explore regions of the conformational space that are virtually inaccessible to the unbound form. Whereas direct observation of target dynamics has found a significant role for conformational selection, 9] induced-fit effects have been shown to be important in the binding pocket of a ionotropic glutamate receptor. The idea that both mechanisms play a role in ligand/target binding is gaining new ground. Similar to enzymatic catalysis, the interplay between the two mechanisms may be regulated by the different time scales that are involved and by allosteric effects. 13] Herein, we have tested the various hypotheses in the pharmacologically relevant case of the phosphoinositide 3-kinases (PI3K). We performed four long, all-atom molecular dynamics (MD) simulations (each lasting 1 ms or more), extensive bias-exchange metadynamics calculations, and analyzed the conformational space that is spanned by the available crystal structures. The finding that the PI3K-signaling pathway is often deregulated in cancer has fueled an increasing interest in designing selective and potent inhibitors of PI3K kinases 16] There are four different isoforms of class I PI3Ks. These isoforms have distinct substrate specificities and different roles in discrete cellular processes. Given their high sequence retention (95 % or more) and a nearly identical adenosine triphosphate (ATP) binding pocket, it is not surprising that a potent and selective inhibitor for only one of the isoforms had remained elusive for a long time. The first selective inhibitor for the delta isoform was reported in 2003. Recently, the crystal structures of various PI3K isoforms in complexes with different inhibitors were published. These structures showed a significant conformational variability in the binding cavity. This variability is particularly prominent in the oncogenic target PI3K-g, which makes it a valid and interesting system with which to explore conformational selection versus the induced-fit hypothesis (Figure 1; see also Figures S1 and S2 in the Supporting Information). We explored the conformational space of the apo form (1E8Y) and the L64cocrystallized holo form (3IBE) of PI3K-g by performing two fully solvated MD simulations that lasted 1.5 ms and 1 ms, respectively. The dynamics were analyzed by principal component analysis (PCA) of the full MD trajectory and considered first the C a coordinates, and then only the atoms defining the binding pocket (Table S2 in the Supporting Information). The global C a PCA vectors of the apo and the holo forms are similar, as shown by the overlap of their covariance matrices (ca. 0.4). The first two vectors describe functionally relevant, hinge-like motions and the relative rotations of the N-terminal and C-terminal lobe. The local binding pocket vectors describe the opening and closing of the cavity (Figure S3 in the Supporting Information), but the overlap of the covariance matrices of the apo and the holo forms is smaller (ca. 0.2). The free energy surfaces (FESs) shown in Figure 2 are obtained by projecting MD trajectories of the apo form (Figure 2a, b) and the holo form (Figure 2c,d) on the first two C a coordinates and binding pocket vectors of the apo-form trajectory. The convergence of the FESs of the apo form and the effect of the initial structure was checked by performing an additional 1 ms long MD simulation. This simulation started from a different crystal structure (3DBS) after the removal of the GD9 ligand, and the new FES was compared with the [*] Dr. M. D’Abramo, Dr. F. L. Gervasio Structural Biology and Biocomputing Programme Spanish National Cancer Research Center (CNIO) C/Melchor Fernandez Almagro 3, 28029 Madrid (Spain) E-mail: flgervasio@cnio.es

On the nature of DNA hyperchromic effect.

A combined theoretical-experimental study of the hyperchromic effect as occurring in the denaturation of a double stranded polyA-polyT is presented. Our theoretical/computational procedure allows us to reproduce the essential features of the experimental spectra and to characterize those molecular interactions responsible for the changes in the UV absorbance. We found that although excitonic intrastrand interactions strongly affect the absorbance, they are almost fully maintained in the single-stranded DNA. Our data indicate that hyperchromic effect originates from the higher delocalization of the excitonic states in the denaturated DNA with respect to the double-stranded conformation.

Engineering a Nickase on the Homing Endonuclease I-Dmoi Scaffold.

Background: The use of nickases may avoid toxicity associated with the NHEJ repair pathway in nuclease-induced gene targeting. Results: Decoupling the action of the I-DmoI catalytic residues acting on each strand generates a nickase. Conclusion: An I-DmoI variant was developed for cleaving one of the strands of its DNA target. Significance: This is the first structure of an engineered nickase in an LAGLIDADG scaffold. Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologous recombination, an error-free mechanism, or by non-homologous end joining, a process susceptible to introducing errors in the repaired sequence. The type of DNA cleavage might alter the balance between these two alternatives. The use of “nickases” producing a specific single strand break instead of a double strand break could be an approach to reduce the toxicity associated with non-homologous end joining by promoting the use of homologous recombination to repair the cleavage of a single DNA break. Taking advantage of the sequential DNA cleavage mechanism of I-DmoI LAGLIDADG homing endonuclease, we have developed a new variant that is able to cut preferentially the coding DNA strand, generating a nicked DNA target. Our structural and biochemical analysis shows that by decoupling the action of the catalytic residues acting on each strand we can inhibit one of them while keeping the other functional.

Modeling conformational transitions in kinases by molecular dynamics simulations: achievements, difficulties, and open challenges

Protein kinases work because their flexibility allows to continuously switch from inactive to active form. Despite the large number of structures experimentally determined in such states, the mechanism of their conformational transitions as well as the transition pathways are not easily to capture. In this regard, computational methods can help to shed light on such an issue. However, due to the intrinsic sampling limitations, much efforts have been done to model in a realistic way the conformational changes occurring in protein kinases. In this review we will address the principal biological achievements and structural aspects in studying kinases conformational transitions and will focus on the main challenges related to computational approaches such as molecular modeling and MD simulations.

Role of the hydrophilic spacer of glucosylated amphiphiles included in liposome formulations in the recognition of Concanavalin A.

The functionalization of liposomes with glycosylated amphiphiles is an optimal strategy for targeted drug delivery, leading to enhanced efficacy as well as to reduced side effects of drugs. In fact, the presence of natural or synthetic glycolipids in vesicle formulations might increase their specificity toward lectins, a class of non-enzymatic sugar-binding proteins involved in cellular recognition and adhesion. The capability of a new glucosylated synthetic amphiphile to interact with Concanavalin A (Con A), a plant lectin used as model system, was investigated by a synergic experimental and computational approach, both as pure component and in formulation with a natural phospholipid. The comparison of the affinity with Con A of the new glucosylated amphiphile with respect to that of a previously described structural analogue demonstrates that the hydrophilic spacer length controls the exposure of the glucose residue on liposome surface, and consequently the recognition by the lectin.

Theoretical-computational modeling of photo-induced charge separation spectra and charge recombination kinetics in solution.

In this study we propose a theoretical-computational method, essentially based on molecular dynamics simulations and quantum-chemical calculations, for modelling the photo-induced charge separation (CS) and the subsequent charge recombination (CR) processes in solution. In particular we have reproduced the low-energy UV-Vis spectra of systems composed by an aromatic species (Ar = benzene or indene) and tetracyanoethylene (TCNE) in chloroform solution, dominated by the formation of the Ar(+)-TCNE(-) ion pair (IP) complex. The kinetics of the charge recombination process leading to the regeneration of Ar and TCNE has also been modelled. In both the cases the agreement with the experimental data is satisfactory. Although the presence of systematic deficiencies makes our approach unable to address some key aspects of the above processes (e.g. the ultrafast internal vibrational redistribution), it appears to be a rather promising tool for modelling the CS-CR process for atomic-molecular systems of very high complexity. The involvement of the triplet IP complex has also been discussed.