Activity and structural dynamics of human ABCA1 in a lipid membrane

The eukaryotic Plasma Membrane (PM) houses thousands of Membrane Proteins (MPs), which perform countless necessary functions for cellular survival and proliferation 1. Importantly, these MPs reside within the context and environment of the PM and require the surrounding lipid bilayer to maintain their native, functionally active conformations. ATP-Binding Cassette (ABC) transporters are canonical MPs that mediate the passage of various substances across the lipid bilayer 2, 3. They do so by coupling the movement of substrates through the transmembrane domain (TMD; i.e., lipids, drugs, sterols) to the binding and hydrolysis of adenosine triphosphate (ATP) 4, 5, 6. ATP binds at the interface of two cytosolic nucleotide binding domains (NBDs); closure of NBDs propagates structural alterations to the TMDs which change conformation to mediate the passage of the substrate across the bilayer. The allosteric linkage between the TMDs and NBDs requires interfacial helices and likely depends on native TMD-lipid interactions 7.

One particularly challenging class of substrates for passage across the PM are the components which comprise the lipid bilayer itself. The eukaryotic plasma membrane is composed of a variety of structural lipids, including those with phosphatidyl-choline (PC), phosphatidyl-ethanolamine (PE), and phosphatidyl-serine (PS), among others 8. Maintenance of this asymmetric PM is required for cell survival, and phospholipid trafficking deficiencies are a hallmark of several cancers 9. In addition to phospholipids, the PM of human cells contains approximately 30% cholesterol – cholesterol trafficking is particularly critical as this sterol affects the local bilayer rigidity and phospholipid/MP packing 8. For cholesterol shuttling between cells and tissues, it must first be extracted from the PM: sterol removal from lipid membranes requires overcoming the immense energetic barrier of pulling this highly insoluble moiety from the hydrophobic core of the bilayer 10. However, this process occurs readily within the human body, as the flux of cholesterol throughout tissues is quite high (i.e., 10 mg/day/kg) – aberrant cholesterol efflux and metabolism are associated with poor atherosclerotic health, along with cancer 11, 12, 13. The mechanistic pathway for the removal of cholesterol from the PM for subsequent secretion from the body is termed the reverse cholesterol transport (RCT) pathway in which the ABC transporter ABCA1 plays a central role 14.

After the initial discovery of the abca1 gene, ABCA1 protein expression was quickly found to be inversely correlated with cellular cholesterol levels 6, 15, 16. It has been well established through cell-biology based characterization that ABCA1 mediates the efflux of cholesterol and phospholipids with PC headgroups from the membrane to the extracellular acceptor protein, apolipoprotein A-I (apoA-I) 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. Together with apoA-I, sterols and phospholipids form nascent high-density lipoprotein (nHDL). ABCA1 and apoA-I are vital in the RCT as they mediate sterol efflux from macrophages, which is essential for clearance of cardiovascular plaques; therefore ABCA1 function is directly linked to atherosclerosis 28, 29. Genetic mutations to the abca1 gene are naturally occurring and are associated with Tangier’s Disease, which results in impaired cholesterol clearance 30, 31. ABCA1 function has further been implicated in neurodegenerative disorders, such as Alzheimer’s Disease 32. ABCA1 has long been suggested as a potential drug target 30, 31, 33. Thus, elucidating how the ABCA1 TMD drives lipid export and how this process can be pharmacologically regulated is of immense biomedical importance.

The overall domain architecture of ABCA1 was elucidated by a previously reported cryo-EM structure of ABCA1 in detergent 34 . While native MPs reside within the complex PM, detergents are a common lipid mimetic utilized for solubilization and biochemical characterization of MPs 35. Detergents are known to affect the biochemical activity of MPs, which seems particularly true for detergent-solubilized ABC transporters as a variety of deleterious effects on ATP-hydrolysis activities have been reported in the literature 37, 41, 35, 36. In line with this notion, the ABCA1 protein in the detergent digitonin used for previous structural studies exhibited minimal ATP hydrolysis activity 34. In addition to the TMDs and NBDs which are present in all ABC transporters, ABCA1 contains a strikingly large extra-cellular domain (ECD) which interacts with apoA-I 19, 21, 22, 34. The ECD contains approximately 850 amino acid residues and its pinnacle reaches 100 Å above the membrane plane. Naturally occurring mutations in the ABCA1 ECD have shown impaired apoA-I-dependent cholesterol efflux 24, 37. However, the exact function of the ECD and how this activity is coupled to the TMDs and NBDs to coordinate lipid movement from the membrane bilayer to apoA-I are unknown.

One notable limitation in previous ABCA1 investigations is that various studies have used protein from distinct sources and in different membrane environments, thus preventing reliable correlation of the multiple activities and dynamic conformations of ABCA1. To overcome this technical hurdle, we have established an inducible ABCA1 expression system and a unified workflow of characterizing ABCA1 in nanodiscs containing a defined lipid system. Taking advantage of this platform, we utilized a combination of cell-based, biochemical, structural, and computational approaches to gain mechanistic insights into ABCA1 functions.

留言 (0)

沒有登入
gif