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.
