Neurological drugs that struggle to cross the blood-brain barrier could fit in the hold of a large facilitator superfamily domain of 2A (MFSD2A) – a kind of molecular ferry. MFSD2A normally carries omega-3 fatty acids into the brain. But now that Columbia University scientists have determined the three-dimensional structure of MFSD2A, they hope drugs can be developed that are compatible with the van’s specifications. For drugs like that, the impassable would be, well, passable.
The three-dimensional structure, which was determined by means of single-particle cryo-electron microscopy, shows how omega-3 fatty acids bind to the transporter. “This information could enable the development of drugs that mimick omega-3 fatty acids to hijack this system and get into the brain,” said Rosemary J. Cater, PhD, research fellow in the Columbia University laboratory, led by Filippo Mancia, PhD, and Associate Professor of Physiology and Cellular Biophysics.
Cater is the lead author and Mancia is the senior author of a study (“Structural basis of omega-3 fatty acid transport across the blood-brain barrier”) published June 16 in the journal Nature. According to this study, MFSD2A has 12 transmembrane helices that are divided into two pseudosymmetric domains.
“The transporter is in an inward-facing conformation and has a large amphipathic cavity that contains the Na + binding site and a bound lysolipid substrate, which we have confirmed with native mass spectrometry,” write the authors of the article. “Together with our functional analyzes and molecular dynamics simulations, this structure reveals details about how MFSD2A interacts with substrates and how Na + -dependent conformational changes enable these substrates to be released into the membrane through a side gate.”
These results, according to the authors, “could provide a basis for the structure-based design of neurotherapeutic agents that hijack MFSD2A for transport across the blood-brain barrier, which is currently a major bottleneck in neurotherapeutic development”.
The blood-brain barrier is a layer of tightly packed cells that lines the blood vessels of the brain and is eager to prevent toxins, pathogens, and some nutrients from entering the brain. Unfortunately, the layer also blocks many drugs that are otherwise promising candidates for treating neurological diseases.
Essential nutrients such as omega-3 fatty acids require the support of special transport proteins such as MFSD2A. “The vans are like bouncers in a club who only let in molecules with invitations or backstage passages,” explains Cater. “If we understand what MFSD2A looks like and how omega-3s are drawn across the blood-brain barrier, we can get the information we need to develop drugs that can trick that bouncer and get tickets.”
To visualize MFSD2A, Cater used a technique called single particle cryo-electron microscopy (cryo-EM).
“The nice thing about this technology is that we can see the shape of the van with details down to a fraction of a billionth of a meter,” said Mancia. “This information is crucial to understand how the transporter works at the molecular level.”
For cryo-EM analysis, protein molecules are suspended in a thin layer of ice under an electron microscope. Powerful cameras take millions of images of the proteins from countless angles, which can then be put together to form a 3D map.
On this map, researchers can create a 3D model of the protein and put each atom in its place. “It reminds me of solving a puzzle,” noted Mancia. This technique has become remarkably powerful in visualizing biological molecules in recent years, thanks in part to Joachim Frank, PhD, Professor of Biochemistry and Molecular Biophysics at Columbia University’s Vagelos College of Physicians and Surgeons, who won the 2017 Nobel Prize for his role in development of cryo-EM data analysis algorithms.
“Our structure shows that MFSD2A has a bowl-like shape and that omega-3 fatty acids bind to a specific side of that bowl,” explained Cater. “The shell is upside down and facing the inside of the cell, but this is just a single 3D snapshot of the protein that has to move in real life to transport the omega-3 fatty acids. To understand exactly how it works, we either need several different snapshots or, even better, a film of the transporter in motion. “
MFSD2A models show how the molecule transports omega-3 fatty acids and other lipids to the brain. Here two snapshots of MFSD2A show two lipids – LPC 18: 3 (left) and Omega-3 (right) – in the intracellular cavity of the transporter. [Rosemary J. Cater, PhD, Columbia University]To understand what these movements might look like, a second co-lead on the study, George Khelashvili, PhD, assistant professor of physiology and biophysics at Weill Cornell Medicine, used the 3-D model of the protein as a starting point to run computer simulations that revealed how the van moves and adjusts its shape to release omega-3 fatty acids into the brain.
A third co-leader of the study, David Silver, PhD, professor at Duke-NUS Medical School in Singapore and pioneer of MFSD2A biology, together with his team tested and confirmed hypotheses derived from the structure and computer simulations about the functioning of MFSD2A have been derived to locate certain parts of the protein that are important.
The team also included researchers from the New York Structural Biology Center, the University of Chicago, and the University of Arizona, all of whom used their specific skills to make this project possible.
The team is now investigating how the transporter recognizes omega-3 fatty acids from the bloodstream for the first time. “But our study has already given us tremendous insight into how MFSD2A supplies omega-3s to the brain,” noted Cater. “We’re really excited to see where our results will lead.”