FRIDaY

Increasing attention is being focused on the role of protein structural dynamics in crucial cellular signaling pathways and modulating structural dynamics is becoming an important avenue of exploitation for the discovery of new therapeutic compounds. However, there remains a serious paucity of experimental techniques that permit one to obtain relevant data related to structural dynamics on appropriate timescales. New approaches are needed to both elucidate and measure physical properties directly related to structural dynamics. Various physical chemical methods have been used to probe the structural dynamics, but each technique has it own limitations. So the search for new methods, complementary to existing ones, is still underway. In this proposal, our primary objective is to develop THz/far infrared absorption spectroscopy for the ligand binding events. Ligand binding will influence the underlying collective motions in this frequency range, which can be captured by far-IR experiments and interpreted through molecular simulations and structural analysis. Previously, we combined far-IR and molecular dynamics simulations to study the response of a PDZ domain to the binding of a small peptide ligand to elucidate the mechanism of allostery. We showed that exploitable information concerning changes in low frequency collective motions could be obtained even for proteins where there is no substantial conformational change upon ligand binding. This integrated approach allowed us to quantify a mechanism of allostery in a PDZ domain. In this project, we aim to enlarge the field of application and characterize the allosteric behavior in nuclear receptor (NR) proteins, which constitute a superfamily of proteins that function as DNA-binding, ligand dependent transcription factors. Being a larger, more complex protein than the one used in our preliminary study, they are implicated in the transcriptional cascade underlying many physiological phenomena making them one of the major signal transduction paradigms in metazoans. Indeed, evidence suggests that there exist multiple mechanisms exploiting structural dynamics and allostery that implicate ligand, DNA, co-activator and co-repressor binding, as well as post-translational modifications. Central to these mechanisms is the ligand binding domain (LBD), which acts as an allosteric hub, transmitting binding events to other protein interfaces and domains. We will focus on Peroxisome Proliferator-Activated Receptor gamma (PPAR-gamma) and its heterodimeric partner, RXRa, a nuclear receptor complex that is a particularly important target for development of therapeutic compounds for multiple diseases, including diabetes and cancer. Our project will begin with a study of the LDBs in both wild-type and mutant forms. We will characterize the effects of ligands, including agonists, antagonists and co-regulator peptides by far infrared absorption spectroscopy, molecular dynamics simulations and biophysical/structural characterization and interpret the results in the context of allosteric effects. Following the study of the LDB, we will expand our study towards heterodimer structures. A second objective is to gain molecular insight into the mechanisms of selective modulation of NRs activity by small-molecule ligands and ultimately make the link with their pharmacological profile. The consortium is comprised of members complementary in their expertise coming from fields of physical and computational chemistry, biophysics and structural biology. Through the fundamental research to be carried out by this high-level multi-disciplinary unit, a novel approach for the study of ligand binding in biomolecular systems will emerge. This, we believe, makes it a completely original methodological approach that we plan to further develop in the context of this call.