Protein-protein docking using geometrical arguments

This project aims to develop nobels algorithms to model protein-protein complexes, a very important aspect in biophysics. The algorithm presented based only on geometrical arguments, is intended to be a first and fast approach to get the most probable configurations. The algorithm finds the best positions producing only a small number of solutions (over 250 solutions). The method is based on 2D FFT (fast fourier transform) and orthographic projections of the proteins. The method allows us to find solutions around 15 Ǻ of Cα root mean square deviation for proteins with low electrostatic interactions

New Monte Carlo Based Technique To Study DNA–Ligand Interactions

We present a new all-atom Monte Carlo technique capable of performing quick and accurate DNA–ligand conformational sampling. In particular, and using the PELE software as a frame, we have introduced an additional force field, an implicit solvent, and an anisotropic network model to effectively map the DNA energy landscape. With these additions, we successfully generated DNA conformations for a test set composed of six DNA fragments of A-DNA and B-DNA. Moreover, trajectories generated for cisplatin and its hydrolysis products identified the best interacting compound and binding site, producing analogous results to microsecond molecular dynamics simulations. Furthermore, a combination of the Monte Carlo trajectories with Markov State Models produced noncovalent binding free energies in good agreement with the published molecular dynamics results, at a significantly lower computational cost. Overall our approach will allow a quick but accurate sampling of DNA–ligand interactions.The authors thank the Barcelona Supercomputing Center for computational resources. This work was supported by grants from the European Research Council—2009-Adg25027-PELE European project and the Spanish Ministry of Economy and Competitiveness CTQ2013-48287 and “Juan de la Cierva” to F.L.Peer ReviewedPostprint (author's final draft

Ecoupling server: A tool to compute and analyze electronic couplings

Electron transfer processes are often studied through the evaluation and analysis of the electronic coupling (EC). Since most standard QM codes do not provide readily such a measure, additional, and user-friendly tools to compute and analyze electronic coupling from external wave functions will be of high value. The first server to provide a friendly interface for evaluation and analysis of electronic couplings under two different approximations (FDC and GMH) is presented in this communication. Ecoupling server accepts inputs from common QM and QM/MM software and provides useful plots to understand and analyze the results easily. The web server has been implemented in CGI-python using Apache and it is accessible at http://ecouplingserver.bsc.es. Ecoupling server is free and open to all users without login.This work has been funded by the Spanish Ministry of Education and Science through the Project CTQ2013-48287.Peer ReviewedPostprint (author's final draft

Mapping biophysics through enhanced Monte Carlo techniques

This thesis is focused on the study of molecular interactions at the atomistic detail and is divided into one introductory chapter and four chapters referencing different problems and methodological approaches. All of them are focused on the development and improvement of computational Monte Carlo algorithms to study, in an efficient manner, the behavior of these systems at a classical molecular mechanics level. The four biophysical problems studied in this thesis are: induced fit docking between protein-ligand and between DNA-ligand to understand the binding mechanism, protein stretching response, and generation/ scoring of protein-protein docking poses. The thesis is organized as follows: First chapter corresponds to the state of the art in computational methods to study biophysical interactions, which is the starting point of this thesis. Our in-house PELE algorithm and the main standard methods such as molecular dynamics will be explained in detail. Chapter two is focused on the main PELE modifications to add new features, such as the addition of a new force field, implicit solvent and an anisotropic network specific for DNA simulation studies. We study, compare and validate the conformations generated by six representative DNA fragments with the new PELE features using molecular dynamics as a reference. Chapter three is devoted to applying the new methods implemented and tested in PELE to study protein-ligand interactions and DNA-ligand interactions using four systems. First, we study the porphyrin binding to Gun4 protein combining PELE and molecular dynamics simulations. Besides, we provide a docking pose that has been corroborated by a new crystal structure published during the revision process of the submitted study showing the accuracy of our predictions. In the second project, we use our improved version of PELE to generate the first structural model of an alpha glucose 1,6-bisphosphate substrate bound to the human Phosphomannomutase 2 demonstrating that this ligand can adopt two low-energy orientations. The third project is the study of DNA-ligand interactions for three cisplatin drugs where we evaluate the binding free energy using Markov state models. We show excellent results respect another free energy methods studied with molecular dynamics. The last project is the study of the daunomycin DNA intercalator where we simulate and study the binding process with PELE. Chapter four is focused on the computational study of force extension profiles during the protein unfolding. We added a dynamic harmonic constraint following a similar procedure applied in steered molecular dynamics to our Monte Carlo approach to fix or pull some selected atoms forcing the protein unfolding in a defined direction. We implement and compare with steered molecular dynamics this technique with Ubiquitin and Azurin proteins. Moreover, we add this feature to a well-known algorithm called MCPRO from William Jorgensen¿s group at YALE University to evaluate the free energy associated to the unfolding of the deca-alanine system. Chapter five corresponds to the introduction of a multiscale approach to study protein-protein docking. A coarse-grained model will be combined with a Monte Carlo exploration reducing the degrees of freedom to generate thousands of protein-protein poses in a quick way. Poses produced by this procedure will be refined and ranked through a protonation, hydrogen bond optimization, and minimization protocol at the all-atom representation to identify the best poses. I present two test cases where this procedure has been applied showing a good accuracy in the predictions: tryptogalinin and ferredoxin/flavodoxin systems.Aquesta tesi es centra en l'estudi de les interaccions moleculars amb detall atomic i es divideix en un capítol d'introducció i quatre capítols que fan referència a diferents problemes i enfocaments metodològics. Tots ells se centren en el desenvolupament i millora dels algoritmes computacionals de Monte Carlo per estudiar, de manera eficient, el comportament d'aquests sistemes a un nivell mecànica molecular clàssica. Els quatre problemes biofísics estudiats en aquesta tesi són: acoblament induït entre la proteïna-lligand i entre DNA-lligant per comprendre el mecanisme d'unió, resposta de les proteïnes a l'estirament, i la generació/puntuació d'acoblament entre poses proteïna-proteïna. La tesi s'organitza de la següent manera: El primer capítol correspon a l'estat de l'art en mètodes computacionals per estudiar les interaccions biofísiques, que és el punt de partida d'aquesta tesi. El nostre PELE algoritme i els principals mètodes estàndard com ara la dinàmica molecular s'explicaran en detall. El capítol dos es centra en les principals modificacions PELE per afegir noves característiques, com ara l'addició d'un nou camp de força, solvent implícit i modes normals per aquests estudis de simulació d'ADN. Es fa un estudi, comparació i validació de les conformacions generades per sis fragments d'ADN representatius amb PELE utilitzant dinàmica molecular com a referència. El tercer capítol està dedicat a l'aplicació dels nous mètodes implementats i provats en PELE per estudiar les interaccions proteïna-lligand i la interacció lligand-DNA utilitzant quatre sistemes. En primer lloc, se estudia la unió a proteïnes GUN4 combinant PELE i simulacions de dinàmica molecular. A més, es proposa un acoblament que ha sigut corroborat per una nova estructura cristal·lina publicada durant el procés de revisió de l'estudi mostrant l'exactitud de les nostres prediccions. En el segon projecte, hem utilitzat la nostra versió millorada de PELE per generar el primer model estructural d'una glucosa alfa substrat 1,6-bisfosfat unit a la fosfomanomutasa humana 2, que demostra que aquest lligant pot adoptar dues orientacions de baiza energia. El tercer projecte és l'estudi de les interaccions d'ADN lligant per tres medicaments cisplatí on se avalua l'energia lliure d'unió utilitzant Markov States Models. Es mostren excel·lents resultats respecte d'altres mètodes d'energia lliure estudiats amb dinàmica molecular. L'últim projecte és l'estudi de l'intercalador d'ADN anomenat daunomicina on es simula i estudia el procés d'unió amb PELE. El capítol 4 es centra en l'estudi computacional dels perfils d'extensió de la força durant el desplegament de la proteïna. Hem afegit una restricció harmònica dinàmica seguint un procediment similar al aplicat en dinàmica molecular en el nostre algoritme Monte Carlo per fixar o moure alguns àtoms seleccionats obligant a desplegar la proteïna en una direcció definida. Aquesta tècnica s'ha implementat i comparat amb dinàmica molecular per les proteïnes ubiquitina i azurin. D'altra banda, hem afegit aquesta modificació a un algoritme ben conegut anomenat MCPRO del grup de William Jorgensen a la Universitat de Yale per avaluar l'energia lliure associada al desplegament del sistema deca alanina. El capítol cinc correspon a la introducció d'un enfocament multiescala per estudiar l'acoblament proteïna-proteïna. Un model de gra gruixut es combinat amb una exploració Monte Carlo per reduir els graus de llibertat i generar milers de poses proteïna-proteïna d'una manera ràpida. Les poses produides per aquest procediment es perfeccionan i evaluan a través d'una protonació, optimització d'enllaços d'hidrogen, i minimització a escala atòmica per identificar les millors poses. Es presenten dos casos de prova on s'ha aplicat aquest procediment que mostra una bona precisió en les prediccions: tryptogalinin i ferredoxina / flavodoxina systems

Direct Measurement of the Nanomechanical Stability of a Redox Protein Active Site and Its Dependence upon Metal Binding

The structural basis of the low reorganization energy of cupredoxins has long been debated. These proteins reconcile a conformationally heterogeneous and exposed metal-chelating site with the highly rigid copper center required for efficient electron transfer. Here we combine single-molecule mechanical unfolding experiments with statistical analysis and computer simulations to show that the metal-binding region of apo-azurin is mechanically flexible and that high mechanical stability is imparted by copper binding. The unfolding pathway of the metal site depends on the pulling residue and suggests that partial unfolding of the metal binding site could be facilitated by the physical interaction with certain regions of the redox protein.We are grateful to A. Donaire and I. Díez-Pérez for discussions, and to the Catalan government (grant 2014SGR-1251), the Spanish government (grant CTQ2013-43892R) and the European Research Council (PELE ERC-2009-Adg 25027) for financial support.Peer ReviewedPostprint (author's final draft

Absolute Free Energy of Binding Calculations for Macrophage Migration Inhibitory Factor in Complex with a Druglike Inhibitor

Calculation of the absolute free energy of binding (ΔGbind) for a complex in solution is challenging owing to the need for adequate configurational sampling and an accurate energetic description, typically with a force field (FF). In this study, Monte Carlo (MC) simulations with improved side-chain and backbone sampling are used to assess ΔGbind for the complex of a druglike inhibitor (MIF180) with the protein macrophage migration inhibitory factor (MIF) using free energy perturbation (FEP) calculations. For comparison, molecular dynamics (MD) simulations were employed as an alternative sampling method for the same system. With the OPLS-AA/M FF and CM5 atomic charges for the inhibitor, the ΔGbind results from the MC/FEP and MD/FEP simulations, −8.80 ± 0.74 and −8.46 ± 0.85 kcal/mol, agree well with each other and with the experimental value of −8.98 ± 0.28 kcal/mol. The convergence of the results and analysis of the trajectories indicate that sufficient sampling was achieved for both approaches. Repeating the MD/FEP calculations using current versions of the CHARMM and AMBER FFs led to a 6 kcal/mol range of computed ΔGbind. These results show that calculation of accurate ΔGbind for large ligands is both feasible and numerically equivalent, within error limits, using either methodology

Porphyrin Binding to Gun4 protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

In oxygenic phototrophs, chlorophylls, hemes and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated and an important regulatory role is attributed to Mgchelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the Mg-chelatase activity but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for Mgprotoporphyrin IX. It revealed that the orientation of 6/7 loop is critical for the binding and the magnesium ion held within the porphyrin is coordinated by Asn211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in Mg-chelatase assay showing that tight porphyrin-binding in Gun4 facilitates its interaction with the Mg-chelatase ChlH subunit. Finally, we introduced the W192A mutation into Synechocystis 6803 cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.This work was supported by project P501/12/G055 of the Czech Science Foundation, and by the National Programme of Sustainability I (LO1416) and by ERC 2009-Adg25027-PELE (to V.G). J.K. was supported by project Algain (EE2.3.30.0059). N.B.P.A., P.A.D., A.A.B. and C.N.H. thank the Biotechnology and Biological Sciences Research Council (BBSRC) U.K. for funding, under award numbers BB/G021546/1 and BB/M000265/1. CNH was also supported by an Advanced Award 338895 from the European Research Council.Peer ReviewedPostprint (author's final draft

Protein-protein docking using geometrical arguments

This project aims to develop nobels algorithms to model protein-protein complexes, a very important aspect in biophysics. The algorithm presented based only on geometrical arguments, is intended to be a first and fast approach to get the most probable configurations. The algorithm finds the best positions producing only a small number of solutions (over 250 solutions). The method is based on 2D FFT (fast fourier transform) and orthographic projections of the proteins. The method allows us to find solutions around 15 Ǻ of Cα root mean square deviation for proteins with low electrostatic interactions

Protein-protein docking using geometrical arguments

This project aims to develop nobels algorithms to model protein-protein complexes, a very important aspect in biophysics. The algorithm presented based only on geometrical arguments, is intended to be a first and fast approach to get the most probable configurations. The algorithm finds the best positions producing only a small number of solutions (over 250 solutions). The method is based on 2D FFT (fast fourier transform) and orthographic projections of the proteins. The method allows us to find solutions around 15 Ǻ of Cα root mean square deviation for proteins with low electrostatic interactions

Ecoupling server: A tool to compute and analyze electronic couplings

Electron transfer processes are often studied through the evaluation and analysis of the electronic coupling (EC). Since most standard QM codes do not provide readily such a measure, additional, and user-friendly tools to compute and analyze electronic coupling from external wave functions will be of high value. The first server to provide a friendly interface for evaluation and analysis of electronic couplings under two different approximations (FDC and GMH) is presented in this communication. Ecoupling server accepts inputs from common QM and QM/MM software and provides useful plots to understand and analyze the results easily. The web server has been implemented in CGI-python using Apache and it is accessible at http://ecouplingserver.bsc.es. Ecoupling server is free and open to all users without login.This work has been funded by the Spanish Ministry of Education and Science through the Project CTQ2013-48287.Peer Reviewe