Molecular Dynamic Studies of Nuclear Receptors Ligand Binding Domain

Nuclear Receptors (NR) function as transcription factors that regulate genes that affect processes like reproduction, development and metabolism. The NRs are activated upon a given signal, which can be a ligand binding or a chemical modification. When activated, the receptors perform a conformational change that opens up interaction surfaces on the ligand binding domain, where coactivators can bind and transcription of the target gene can start. In this thesis the key events for NR activation, the ligand binding/unbinding mechanism and interactions with cofactors, are studied. Furthermore the communication between the ligand binding pocket and the cofactor interaction surface was investigated. To gain insight on the ligand unbinding mechanism we performed molecular dynamics (MD) studies on two NRs. The unbinding mechanism from Retinoic Acid Receptor (RAR) and Estrogen Receptor (ER) was studied with modified MD methods, random acceleration MD and steered MD. In the RAR study 4 unbinding pathways of the ligand retinoic acid were obtained, where one of the pathways were more likely than the others. Thus ligand binding could be obtained without major conformational changes on the receptor structure. In the ER study, three different ligands unbinding from ER α and β was studied. The results showed that an ER agonist or selective agonist could unbind from the receptor without causing major conformational changes, while a slightly more bulky antagonist could not. Thus NR agonist and antagonist would use different unbinding mechanisms. The results from the ER simulations also showed variance in pathway preference between the different ligands. Differences between the ligands and receptor subtypes might therefore also effect the unbinding and hence influence ligand selectivity. When the NR is activated an interaction surface becomes available and cofactors with a conserved motif can bind. In MD studies of Liver Receptor Homologue 1 (LRH-1) and Liver X Receptor (LXR), the interaction between different cofactor peptides and the receptors were characterized. In the LRH-1 study, a specific interaction from an aspartate to the motif was identified while the interactions between LXR and cofactor peptides showed a less specific binding. Thus specificity between LXR-cofactors should be found in other factors. Cofactor interactions were also studied in the context of ligand binding. For LRH-1, a bound ligand to the receptor caused different effects on the receptorcofactor peptides interaction. This indicates a communication pathway between the ligand and the cofactor peptide, an allosteric communication. Allosteric signaling is difficult to study, to do so we used a modified MD technique, anisotropic thermal diffusion method. With this method we were able to identify an allosteric signaling pathway from a coactivator peptide through LXR to the ligand in the ligand binding pocket. LIST OF PUBLICATIONS I. Carlsson, P., Burendahl, S. and Nilsson, L. , Exploring the Unbinding of Retinoic Acid from the Retinoic Acid Receptor using Random Expulsion Molecular Dynamics, 2006, Biophys. J., 91: 3151-3161 II. Burendahl, S. , Danciulescu, C. and Nilsson, L. Ligand unbinding from the estrogen receptor: A computational study of pathways and ligand specificity, 2009, submitted manuscript III. Burendahl, S. , Treuter, E. , and Nilsson, L. Molecular Dynamics simulation of human LRH-1: the impact of ligand binding in a constitutively active nuclear receptor, 2008, Biochemistry, 47(18):