Cells Put Their Best Face Outward

Structure and function of the flippase enzyme.

This dates me a little, but when I was in grad school, the fluid bilayer hypothesis of membrane lipids was still new and exciting. Now canonical, it proposed that cellular membranes have no more structure than a soap bubble, being flat fluids of phospholipids that self-organize into a bilayer with two leaflets, each leaflet keeping its polar or charged head groups out towards aqueous solution, and their lipid tails on the inside, facing the complementary leaflet. At our scale, it seems shockingly fragile and structure-less. But at the micro scale, it is a pretty tough affair. Typical membranes are about 5 nm thick, which seems negligible, but it takes a protein at least 7 alpha helical turns, or 25 amino acids, to span it. Given that the fatty tail length is freely adjustable, as is the chemical nature and charge of the head groups, evolution has evidently optimized the thickness of membranes to provide an optimal tradeoff of structure and lightness. They are tougher than they look.

In this microscopic technique, cells are frozen and cleaved sideways, causing some of the membranes to split along their inner leaflet boundaries. This highlights the proteins and other material embedded within them. Note at the top that a small portion of the plasma membrane of this cell has a quasi-crystalline raft of proteins- a sign of active signalling taking place.

Membranes are also chemically tough, impervious to charged molecules due to their fatty interior. These features made membranes incredibly successful- one of the key foundations of life. Eukaryotes developed a whole second frontier of membranes, as internal organelles like the nucleus, endoplasmic reticulum, golgi, lysozome, and mitochondria. Mitochondria particularly use the imperviousness of membranes to set up complex charge and chemical asymmetries, to serve as batteries, storing up electromotive force from respiration of food and using it to synthesize ATP.

But it turns out that there are some forms of structure amid all the fluidity of the fluid bilayer. There are the proteins, of course, which can organize into crystalline rafts, or hook onto cell walls (in plants and bacteria) or cytoskeletal supports to enforce overall cell shape. There are features of composition that can make membranes more stiff, such as using more rigid, more saturated lipid tails, or having more cholesterol, which serves as a plate-like stiffener. And it also turns out that the two sides of membranes can have markedly different compositions, another indication of just how stable and tough these tiny structures are.

A recent paper revealed the structure of an enzyme (flippase) that helps to enforce the asymmetry of composition between the inner and outer leaflets of eukaryotic plasma membranes. Why would such asymmetry exist? The reasons are not all clear, really. One aspect is the charge imbalance, whereby the inner (cytoplasmic) leaflet has more heavily charged phospholipids. There could also be defense issues, particularly among bacterial, which might want to present certain lipid head groups externally, and use other ones internally. Another is signaling, where certain phospholipids are chemically modified to serve as protein attachments and other forms of signaling, and thus need to be on the correct side of the wall. One prominent example is phosphotidylserine, which is usually kept on the inner leaflet. During cell suicide, (apoptosis), however, the (flippase) enzymes that keep it there are cleaved and disabled, while other enzymes (scramblases) that degrade the membrane composition asymmetry are activated, causing phosphatidylserine to be shown on the cell's outside, which is in turn a signal to traveling macrophages to attack and eat that cell.

So flippases spend their lives scavanging phophotidylserine from the outer membrane leaflet and transferring it to the interior leaflet, constituting one sign to the outside that yes, I am still alive. The process violates the concentration gradient of phosphatidylserine, so needs energy, which comes in as ATP. We end up with a rather complex two protein system that itself has to be consistently oriented the right way in the plasma membrane, cleaves ATP, phosphorylates itself briefly, grabs phosphatidylserine specifically from the outer leaflet of the membrane, and then transports it across to the inner membrane.

This schematic illustrates the enzymatic cycle. The phosphatidylserine to be transported is at bottom, in green, on the external face of the membrane. A complex ATP=>ADP cycle dramatically alters the shape of the top of the enzyme on the cytoplasmic face, which at the E2P step is propagated down to a gap which opens between the two proteins- the portions colored purple and beige, which are situated in the membrane. This lets a phosphatidylserine to slip into a pocket that binds it selectively, after which the phosphate leaves the upper part, the enzyme recloses, and the phosphatidylserine is released to the other face of the membrane.

This structure was arrived at with the new techniques of electron microscopy that have allowed protein structures to be determined without crystallization, a development that has been particularly beneficial for membrane proteins that tend to be very hard to crystallize. The project also used a series of ATP and phosphatidylserine analogs that helped freeze the proteins in certain conformations through the reaction cycle, providing the data that informs the model above.

A closeup of the phosphatidylserine binding site, the lipid tails pointing upward. Ther are numerous amino acid side chains from the protein (such as asparagine (N) 353, serine (S) 358, etc. that coordinate the phosphatidylserine specifically, making this a transporter almost exclusively for this phospholipid alone. Other hydrophobic side chains such as phenylalanine (F) 107 and 368 form congenial interactions with the lipid tails.

Binding of phosphatidylserine is specific, but it can not be very strong, since the point of the reaction cycle is to release it again rapidly. Once binding has established specificity, it induces dephosphorylation, which then induces further conformation changes that lock the outward access of the phospholipid and destabilize its binding to the protein.

A cross-section of the full structure (right), and schematic showing (left) the series of structural elements of the two proteins of the transporter (CDC50A, now called TMEM30A in red, and ATPA1, the ATPase, in all the other colors.) The full structure (with no phospholipid or ATP present) has the ATPase on a large domain sticking out into the cytoplasm, and the key phosphatidylserine binding cleft (between the purple and beige sections, buried in the membrane.

It is wonderful to live in an age when such secrets of life, once utterly unsuspected, and then veiled in unreachable technical obscurity, are revealed in mechanistic detail.

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