Dottorato in BIOCHIMICA

Titolo della tesi: Deciphering the activation mechanism of Sigma 1 Receptor and the nature of its endogenous ligands

The Sigma-1 receptor protein (S1R) is a transmembrane protein resident in the endoplasmic reticulum, involved in various molecular mechanisms, including those related to neurodegenerative diseases such as Alzheimer’s disease, Huntington's disease, Parkinson’s disease and Amyotrophic Lateral Sclerosis’s disease. The neuroprotective role of S1R has led to increasing pharmacological interest. Significant advances have been made in understanding the biological role of the protein, and structural biology has made a strong contribution by solving crystallographic structures in complex with some molecules identified as agonists and antagonists of hS1R. However, despite the progress made in recent years, some of the most important hS1R features, required to fully understand its function namely the receptor's endogenous ligand(s), the molecular mechanism of ligand access to the binding site, and the oligomerization mechanism influenced by agonists and antagonists have not yet been unequivocally determined. Using computational techniques such as molecular dynamics and virtual screening, and experimental techniques such as cryogenic electron microscopy (Cryo-EM) and fluorescence assays, we aimed at addressing these key characteristics of hS1R. To shed light on the nature of the endogenous hS1R ligand(s), we used a combination of computational virtual screening (VS), electron density maps fitting, and fluorescence titration assays to measure ligand binding to hS1R in vitro. We found that the ligands with the highest affinity for the receptor were molecules with a steroid motif, and among them, 16,17-didehydroprogesterone was shown by fluorescence titration to bind hS1R with significantly higher affinity than the prototypical hS1R ligand pridopidine in the same assay. Through molecular dynamics simulations, we investigated the mechanism of ligand entry into the binding site. It was hypothesized that ligands access the protein's binding site through a cavity that opens on the surface in contact with the membrane. This was supported by molecular dynamics studies that revealed conformational changes in the hS1R structure, particularly in the membrane-interacting helices, in agreement with previous structural studies on S1R from Xenopus laevis. In parallel, Cryogenic Electron Microscopy (Cryo-EM) was used to study the oligomeric structure of S1R in solution. Cryo-EM particle analysis revealed two main protein states: a trimer and a hexamer. The trimer conserves the same conformation of that observed in X-ray crystallography, whereas in the hexamer the trimers assume different conformations. In particular, the N-terminal α-helices assume a different conformation with respect to the trimer, used to form the oligomeric interface. These results are particularly important since they contribute to disclose the nature of the endogenous ligand, its entry pathway, and the mechanism of oligomerization, which are at the basis of the S1R activation mechanism and are important for the developing of new therapeutic compounds aimed at modulating the receptor in neurodegenerative diseases.