In silico saturation mutagenesis and docking screening for the analysis of protein-ligand interaction: the Endothelial Protein C Receptor case study

BACKGROUND: The design of mutants in protein functional regions, such as the ligand binding sites, is a powerful approach to recognize the determinants of specific protein activities in cellular pathways. For an exhaustive analysis of selected positions of protein structure large scale mutagenesis techniques are often employed, with laborious and time consuming experimental set-up. 'In silico' mutagenesis and screening simulation represents a valid alternative to laboratory methods to drive the 'in vivo' testing toward more focused objectives. RESULTS: We present here a high performance computational procedure for large-scale mutant modelling and subsequent evaluation of the effect on ligand binding affinity. The mutagenesis was performed with a 'saturation' approach, where all 20 natural amino acids were tested in positions involved in ligand binding sites. Each modelled mutant was subjected to molecular docking simulation and stability evaluation. The simulated protein-ligand complexes were screened for their impairment of binding ability based on change of calculated Ki compared to the wild-type. An example of application to the Endothelial Protein C Receptor residues involved in lipid binding is reported. CONCLUSION: The computational pipeline presented in this work is a useful tool for the design of structurally stable mutants with altered affinity for ligand binding, considerably reducing the number of mutants to be experimentally tested. The saturation mutagenesis procedure does not require previous knowledge of functional role of the residues involved and allows extensive exploration of all possible substitutions and their pairwise combinations. Mutants are screened by docking simulation and stability evaluation followed by a rationally driven selection of those presenting the required characteristics. The method can be employed in molecular recognition studies and as a preliminary approach to select models for experimental testing

Modelling the interaction of steroid receptors with endocrine disrupting chemicals

BACKGROUND: The organic polychlorinated compounds like dichlorodiphenyltrichloroethane with its metabolites and polychlorinated biphenyls are a class of highly persistent environmental contaminants. They have been recognized to have detrimental health effects both on wildlife and humans acting as endocrine disrupters due to their ability of mimicking the action of the steroid hormones, and thus interfering with hormone response. There are several experimental evidences that they bind and activate human steroid receptors. However, despite the growing concern about the toxicological activity of endocrine disrupters, molecular data of the interaction of these compounds with biological targets are still lacking. RESULTS: We have used a flexible docking approach to characterize the molecular interaction of seven endocrine disrupting chemicals with estrogen, progesterone and androgen receptors in the ligand-binding domain. All ligands docked in the buried hydrophobic cavity corresponding to the hormone steroid pocket. The interaction was characterized by multiple hydrophobic contacts involving a different number of residues facing the binding pocket, depending on ligands orientation. The EDC ligands did not display a unique binding mode, probably due to their lipophilicity and flexibility, which conferred them a great adaptability into the hydrophobic and large binding pocket of steroid receptors. CONCLUSION: Our results are in agreement with toxicological data on binding and allow to describe a pattern of interactions for a group of ECD to steroid receptors suggesting the requirement of a hydrophobic cavity to accommodate these chlorine carrying compounds. Although the affinity is lower than for hormones, their action can be brought about by a possible synergistic effect

The dissociation of carbon monoxide from hemoglobin intermediate.

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at −25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species

ProCMD: a database and 3D web resource for protein C mutants

Background: Activated Protein C (ProC) is an anticoagulant plasma serine protease which also plays an important role in controlling inflammation and cell proliferation. Several mutations of the gene are associated with phenotypic functional deficiency of protein C, and with the risk of developing venous thrombosis. Structure prediction and computational analysis of the mutants have proven to be a valuable aid in understanding the molecular aspects of clinical thrombophilia. Results: We have built a specialized relational database and a search tool for natural mutants of protein C. It contains 195 entries that include 182 missense and 13 stop mutations. A menu driven search engine allows the user to retrieve stored information for each variant, that include genetic as well as structural data and a multiple alignment highlighting the substituted position. Molecular models of variants can be visualized with interactive tools; PDB coordinates of the models are also available for further analysis. Furthermore, an automatic modelling interface allows the user to generate multiple alignments and 3D models of new variants. Conclusion: ProCMD is an up-to-date interactive mutant database that integrates phenotypical descriptions with functional and structural data obtained by computational approaches. It will be useful in the research and clinical fields to help elucidate the chain of events leading from a molecular defect to the related disease. It is available for academics at the URL http://www.itb.cnr.it/procmd/

Mellom politisk styring og styrt politikk - En diskursanalyse av køprising som klimapolitisk virkemiddel

Denne oppgaven er en diskursanalyse av køprising som klimapolitisk virkemiddel. På tross av at det i dag hersker bred enighet om nødvendigheten av å redusere utslipp av klimagasser, peker mye på et økende gap mellom målsetninger og resultater på området. Samtidig er transport – og særlig veitrafikk – en stor og økende kilde til disse utslippene. Køprising er et virkemiddel som i lengre tid har vært omtalt og anbefalt i fagmiljø tilknyttet transport, og som i perioden mellom 2008 og 2013 ble knyttet opp i mot nasjonale klimamål og statlige løfter om økonomiske midler og samarbeid. Blant byområdene som hadde køprising på agendaen var Trondheim, Kristiansand og Bergen. I empirien inngår tekster knyttet til Framtidens byer, Belønningsordningen og byenes bypakker. Jeg benytter meg av en sosialkonstruktivistisk tilnærming i oppgaven, der jeg anser at det er ulike måter å tolke både klimaproblemet og køprising som virkemiddel på, og at representasjoner av forholdet mellom dem ikke materialiserer seg av seg selv. I oppgaven analyserer jeg tekster knyttet til forvaltningen av en statlig bytransportpolitikk, og sporer forandring i måten forholdet mellom køprising og klima kommer til uttrykk på. Hensikten med oppgaven er å belyse vilkår for interaksjon mellom ulike politiske og administrative myndighetsnivå i skjæringspunktet mellom et klima–, by– og transportpolitisk domene