Structural Basis of Sigma-1 Receptor Ligand Interactions and Signalling

Cells have internal compartments that are specialised for unique functions including energy generation and protein production. The compartments are segregated by internal membranes that are selectively permeable. The membranes contain protein molecules ("membrane proteins") with the critical job of permitting the transfer of matter and information into and out of the intracellular compartments to enable coordination of cellular function. The Sigma-1 Receptor (S1R) is one such protein that is involved in regulating the communication between energy generating mitochondria and the endoplasmic reticulum (ER), which requires energy to produce the proteins necessary for the cell to function effectively. S1R is embedded in the ER membrane of neural cells in a region closely apposed to mitochondria and signals energy demands to the mitochondria. Because of its central role in maintaining an appropriate response to energy demands in neurons, S1R activity impacts a range of neurological conditions. S1R is regulated by both endogenous molecules and exogenous drugs, some of which are currently used therapeutically for the treatment of pain, depression, and schizophrenia. Our work aims to understand the molecular basis of S1R activity in terms of how it interacts with those drugs and how those interactions lead to communication between the ER and mitochondria.
Membrane proteins like S1R that transmit signals across membranes are critical switching points in intracellular communication networks. Because of their functional importance, understanding how these proteins work is important both medically and for biotechnology and industry. As with the macroscopic world, one of the best approaches for understanding how proteins work is by observing them in detail. However, membrane proteins are extremely challenging to study by conventional methods since they must be extracted from the native cellular environment in which they have evolved to be most stable. We have developed methods to produce S1R in large quantities and purify it in large quantities from both bacterial and mammalian cells -- a necessity for robust, atomic-level observation. We propose here to take advantage of the approaches we have developed to study the structure and interactions of S1R in detail. A central technique of the proposed research is Nuclear Magnetic Resonance (NMR). NMR is a flexible, information-rich spectroscopy that provides atomic level information, and is particularly powerful in studying inter-molecular interactions in detail. We anticipate that the results of this study will (i) help to understand the mechanism of S1R in neurological disease, (ii) facilitate design of novel therapeutic agents, and (iii) facilitate structural characterisation of other membrane protein signaling proteins.

The Sigma-1 Receptor (S1R) is a ligand-regulated protein chaperone found at the mitochondria associated ER membrane. Upon activation, S1R binds to the IP3 receptor to regulate inter-organelle calcium signalling. Further activation results in re-localisation of S1R to the plasma membrane where it regulates voltage-gated ion channels. S1R is highly expressed in tissues of the central nervous system, and S1R dysfunction is implicated in pain, amnesia, schizophrenia, depression, stroke, Alzheimer's disease, and addiction. S1R activity is regulated by a large number of drugs, but little is known about the molecular basis of its ligand binding or signalling. No bacterial homologs of S1R have been discovered, and the lack of molecular-level information about S1R function arises largely from the difficulties in producing and stabilising eukaryotic membrane proteins. We have developed a multi-pronged approach to studying S1R structure and interactions in which information from solution NMR studies of bacterially expressed S1R constructs is combined with biophysical and in cell fluorescence studies of mammalian expressed S1R. Details of residues and regions of S1R responsible for ligand and cholesterol binding will be determined by solution NMR on S1R truncation mutants. This information will be validated and extended by studies of full length S1R that is expressed into mammalian cell membranes.

The general areas of impact are to (i) increase biological understanding of a intracellular signaling pathway important for cellular homeostasis, (ii) significantly increase the molecular understanding of ligand interactions in a proven drug target, (iii) extend the use of NMR for studying membrane proteins, and (iii) provide training in the bacterial and mammalian production of membrane proteins, and their study by biophysical and cell biological methods.
The impact of the proposed research will extend beyond the Sigma-1 Receptor (S1R) system that is to be studied, since membrane proteins remain vastly under-represented in the Protein Data Bank (PDB) due to difficulties in production, stabilisation, and crystallisation when solubilised with detergents and lipids. Additional structures will help develop our general understanding of membrane protein structure. Furthermore, the work will expand knowledge about the intermolecular interactions of membrane proteins. Intermolecular interactions of membrane proteins (both protein-protein and protein-small molecule interactions) are highly relevant to many medically important systems, but atomic-level studies of such interactions has been largely absent.
The primary mechanism for communication of this research will be through publication in peer review international journals. Open access publishing options will be used where available. We will liaise at the time of publication with the University of Oxford and MRC press offices to ensure dissemination of results that are of interest to the general public, and take advantage of opportunities to communicate via freely accessible media (such as the Department of Biochemistry and STRUBI websites) in order to extend the impact of the findings. Our results will also be made available on our regularly updated laboratory web sites. Data obtained during this project, such as structural models and NMR chemical shift assignments will be deposited into the open-access Protein Data Bank and BioMagResBank databases, respectively.
Dr. Ortega and PDRA-2 employed on this grant will gain technical skills in manipulation of membrane protein samples and their characterisation by NMR, in-cell fluorescence, and other biophysical methods. In addition they will be trained in writing, IT, and presentational skills, and will benefit from working closely with expert colleagues, thereby enhancing their future research employment prospects.