Major Facilitator Superfamily Domain containing 2A (MFSD2A) is a 58 kDa integral membrane protein that is
highly expressed within endothelial cells of the blood-brain and blood-retinal barriers where it mediates uptake
of the ¿-3 fatty acid docosahexaenoic acid (DHA) into the brain and eyes respectively. DHA comprises up to
20% of total brain lipids. Despite this, the brain cannot synthesise DHA de novo and MFSD2A-mediated transport
is the primary route by which the brain acquires DHA. Mouse MFSD2A KO models exhibit severe brain DHA
deficiency and generalized microcephaly, and human patients with homozygous loss-of-function mutations in
MFSD2A present with severe microcephaly and developmental delay.
Previous cell-based studies have demonstrated that MFSD2A transports DHA in the form of
lysophosphatidylcholine (LPCDHA), but not unesterified fatty acid, in a Na+-dependent manner. Similar to all
other MFS family members, MFSD2A has twelve transmembrane domains but unlike most MFS proteins that
transport water soluble compounds, MFSD2A transports lysolipids, raising the possibility of a unique transport
mechanism. It has been hypothesized that MFSD2A transports mono-acyls chain lipids across membranes by
flipping the LPC between membrane leaflets, acting essentially as a “flippase”. Understanding MFSD2A-
mediated transporter has the potential to aid drug delivery to the brain. Proof of concept studies have indicated
that small molecules covalently linked to LPC can be transported by MFSD2A, potentially providing a new
platform for drug delivery across the blood brain barrier – a major bottleneck in neurotherapeutic development.
Currently, there are no known atomic resolution structures of MFSD2A and a limited understanding of its
molecular transport mechanism. This has hindered our understanding of who DHA is delivered into brain and
precludes structure-based LPC-prodrug design for neurotherapeutic intervention.
Here we present the first structure of MFSD2A, in an apo inward-open conformation - in complex with a Fab to
increase the size of and introduce features to the imaged particles – determined using single particle cryo
electron microscopy to 3.45 Å resolution. In collaboration with, Dr. David Silver at DUKE-NUS (Singapore), a
pioneer in the MSFD2A physiology field, we have developed biochemical assays to probe structure-based
functional hypotheses for MSFD2A in cells, solution, and proteoliposomes. We are proposing to determine the
structures of MSFD2A in complex with its ligand and in an outward-facing conformation, and to use our newly
developed biochemical assays to probe and fully understand the molecular features of these structures. The
results generated from this application will elucidate the molecular-mechanism behind MSFD2A-mediated
LPCDHA transport and will provide information regarding how DHA enters the brain. Furthermore, this research
has the potential to lay the foundation for the rational design of neurotherapeutics that “hijack” MFSD2A for
delivery across the blood brain barrier.