Project Summary/Abstract The bioactive lipid sphingosine-1-phosphate (S1P) plays a key role in regulating the growth, survival and migration of mammalian cells. S1P is produced intracellularly and then released extracellularly to engage in its (patho)physiological roles. The Spinster (Spns) lipid transporters of the major facilitator superfamily (MFS) are critical for transporting S1P across cellular membranes. Of the three Spns proteins in humans, Spns2 functions as the main S1P transporter, which makes it a potential drug target for modulating S1P export and signaling. An endothelial cell-specific defect in Spns2 results in impaired egress of lymphocytes and prevents tumor metastasis in mice, strongly suggesting that Spns2 could be an effective target for reducing metastases by increasing the efficacy of immunotherapy. Thus, detailed characterization of the Spns2 mechanism is of high significance for the development of novel therapeutic strategies for diseases associated with S1P signaling and to target Spns2 as a potential immunosuppressant. The overall goal of this proposal is to define the functional mechanism of the Spns family of sphingolipid transporters. The mechanism of Spns2-mediated S1P transport across cellular membrane remains poorly understood, mainly due to the lack of structural information (Aims 1 and 2). In addition, the precise mechanism of Spns2 regulation is still unclear (Aim 3). We recently defined the proton- dependent conformational dynamics of a bacterial Spns transporter. Our approach capitalizes on a powerful pulsed EPR technique known as Double Electron Electron Resonance (DEER) spectroscopy, an effective nanometer-scale ruler, in the context of high-resolution structures. It is informed by functional studies and contextualized through collaborative molecular modeling. Using this integrated approach, we conduct a thorough mechanistic comparison between human Spns2 and its homologs. The objectives of this proposal are to define the cation- and substrate-coupled conformational cycle of human Spns2 and its bacterial homologs in lipid bilayers. To determine the conformational states involved in the alternating access mechanism, we will apply DEER spectroscopy under conditions expected to stabilize transport intermediates and combine the results with restraint-assisted molecular dynamics to map ligand-coupled conformational changes. Using a similar integrated approach to define the transport mechanism of other Spns family members and their prokaryotic homologs, we will identify the key commonalities and differences in their mechanisms, highlighting the mechanistic flexibility enabling their diverse function with transformative therapeutic potential.