Tag Archives: Phosphatidyl serine


Pickings have been slim lately, but here’s a great paper and a puzzle for you chemists out there. Most chemists (and biologists) know what a lipid bilayer is. It’s basically a soap bubble, with water loving (hydrophilic) groups on the outside of both sides of the bilayer, and hydrocarbon chains within. If the hydrocarbon chains are all stretched out the distance between carbons 1 and 3 is 2.66 Angstroms, and you have an 18 carbon fatty acid (stearic acid) it should be 8 * 2.66 + 1.33 Angstroms long (22.6 Angstroms). Double this for the bilayer and you have a thickness of 45 Angstroms. It’s probably less because carbon chains aren’t extended, partially because of entropy and largely because of cholesterol which breaks up any chance of such order (which maybe an important function for it). Sitting on either side of the lipid bilayer are phosphates esterified to one of the 3 hydroxyls of glycerol, with fatty acids of at least 16 – 18 carbons esterified to the other two. Hanging off the phosphates are a variety of things, but mostly serine and choline, forming phosphatidyl serine (PS) and phosphatidyl choline (PC). Here’s a picture — https://en.wikipedia.org/wiki/Lipid_bilayer.

Scramblases are enzymes which move phospholipids from one side of the lipid bilayer essentially randomizing their composition. They undo the action of other enzymes (called flippases believe it or not) which make the lipid composition of the two leaflets of the lipid bilayer rather different. This isn’t trivial, and is behind an elegant mechanism to show scavenger cells that a cell is dead. FLippases work to put phosphatidyl serine (PS) on the side of the lipid bilayer (the leaflet) facing the cytoplasm. This, of course takes energy, and when a cell lacks energy, entropy takes its course and PS appears on the outer leaflet, telling scavenger cells (phagocytes) to eat (phagocytose) the cell.

So how does an enzyme drag phosphatidyl choline (PC) https://en.wikipedia.org/wiki/Phosphatidylcholine or phosphatidyl serine (PC) across the lipid bilayer — scrambling the compositional asymmetry. Can you figure out a mechanism for a membrane protein to do this without looking at Proc. Natl. Acad. Sci. vol. 113 pp. 140149 – 14054 ’16? Chemists think they’re smart, and if you can design a protein to do this you’re smarter than I am because I’ve always wondered (ineffectually) how this was done for a long time.

The authors describe the structure of a fungal scramblase. It functions as a dimer with each subunit containing a hydrophilic groove containing polar and charged amino acid side chains facing the dimer interface. The protein itself does something unusual — it twists the sheet of the membrane, and decreases the thickness of the membrane from 29 to 18 Angstroms (remember the maximum possible thickness of the lipid bilayer was 45 Angstroms, but isn’t that thick for the reasons given above).

Phosphatidyl choline is a zwitterion (e.g. it contains both negative and positive charges although overall electrically neutral). The charges are separated in space forming a dipole. On the cytoplasmic side of the bilayer the scramblase has some amino acid side chains also forming a dipole, and right near the channel formed by the two hydrophilic grooves of the dimer. So it attracts the head group of PC (phosphate plus choline) as one dipole does to another which is then further attracted to the hydrophilic groove entering it — its hydrocarbon tail remains in the lipid part of the membrane. Then another PC joins the fun, pushing PC #1 farther into the groove, so that a chain of PCs fills the groove, wagging their lipid tails behind them (a la Little Bo Peep).

Clever no?

All is not perfect as the model doesn’t explain how phosphatidyl serine (which isn’t a zwitterion) moves across, but it’s an incredible start.


You are alive because the lipid bilayer of your plasma membrane is asymmetric

You are an organism with trillions of cells. A mosquito bit you depositing millions of viruses in your tissues. The virus can reproduce only within one of your cells and it has exploited all sorts of protein protein chemistry to get in. Antibodies (if you are fortunate enough to have them) can get rid of the extracellular critters. However, 500,000 have made into the same number of your cells, and are merrily trying to reproduce.

How does the asymmetry of the lipid bilayer of your plasma membrane help you survive. If each virus infected cell killed itself before the virus reproduced, you’d survive. Although 500,000 is a large number is is less than 1 millionth of your cell total.

Well you do have intracellular defenses against viruses, called the innate immune system. One of them is a protein with the ugly name of gasdermin D. The activated innate immune system (in the form of inflammatory caspases) cleaves gasdermin. This breaks up the inhibition of the amino terminal part of gasdermin by the carboxy terminal part giving a fragment which binds to one particular membrane component (phosphatidyl serine) which makes up 20% of the inner leaflet of the cell membrane. It then forms a large diameter (to a cell 140 Angstroms is quite large) pore in the cell membrane. No cell can survive this, so it dies, releasing cellular contents (probably some viral components but not fully formed one). For details see [ Nature vol. 535 pp 111 – 116, 153 – 158 ’16 ]

Wait a minute. The toxic gasdermin fragment is also released. So how come it doesn’t kill everything in sight? Because our cellular membranes keep phosphatidyl serine confined to the inner membrane, normal cells don’t show it on their exterior, so they can be bathed in gasdermin with no ill effect. What is responsible for this asymmetry — believe it or not an ATP consuming enzyme called flippase (about this more later) which takes any phosphatidyl serine it finds on the outer leaflet and schleps it back inside the cell.

There is all sorts of elegant chemistry which explains just how gasdermin binds to phosphatidyl serine and none of the many other phospholipids found on the inner leaflet. There is more elegant chemistry explaining how flippase works (see later).

What chemistry cannot explain, is why organisms would ‘want’ an asymmetric membrane. As soon as you get into the function of a particular compound in an organism, chemistry is powerless to tell you why. Nothing else can explain how a given molecule does what it does on the molecular level but that is not enough for a satisfying explanation.

One further explanation before some hard core cellular biochemistry follows (after ***). Our cells are dying all the time. The lining of your gut is replaced every 5 days. Even the longest lasting element of your blood is gone after half a year, and most other elements are turned over at least once a month. When these cells die, they must be cleaned up, without undue fuss (such as inflammation). The cleaners are cells called macrophages. A dying cell releases chemical signals, actually called ‘eat me’, one of which is phosphatidyl serine found on the membrane fragments of a dead cell. The fact that flippases keep it on the inner leaflet means that macrophages won’t attack a normal cell.

Slick isn’t it?


Flippase is a MgATPdependent aminophospholipid translocase. It localizes phosphatidylserine and phosphatidylethanolamine to the inner membrane leaflet by rapidly translocating them from the outer to the inner leaflet against an electrochemical gradient. The stoichiometry between amino phospholipid translocation and ATP hydrolysis is close to one (how will the cell have enough ATP to do anything else?). The flippase is inhibited by high calcium, and by pseudosubstrates such as vanadate, acetylphosphate and para-nitrophenyl phosphate, and by SH reactive reagents such as N-ethylmaleimide and pyridyldithioethylamine (PDA) a specific inhibitor of phospholipid translocation

[ Proc. Natl. Acad. Sci. vol. 109 pp. 1449 – 1454 ’12 ] P4-ATPases are a subfamily of P-type ATPases. They transport aminophospholipids from the exoplasmic to the cytoplasmic leaflet (and are known as flippases). Man has 14 P4-ATPases, expressed in various cell types. They are thought to be similar to the catalytic subunits of the Ca++ ATPase, and the Na, K ATPase, consisting of cytoplasmic, N, P and A domains and a membrane domain made of 10 transmembrane helices (M1 – M10).

[ Proc. Natl. Acad. Sci. vol. 111 pp. E1334 – E1343 ’14 ] The P4-ATPases are thought to resemble the classic P-type ATPase cation pumps — a transmembrane domain of 10 helices and 3 cytoplasmic domains (P for phosphorylation, N for nucleotide binding and A for actuator). ATP8A2 forms an intermediate phosphorylated on aspartic acid (E2P)and undergoes a catalytic cycle similar to the sodium pump (Na+, K+ ATPase). Dephosphorylation of E2P is activated by the transported substrates phosphatidyl serine (PS) and phosphatidyl ethanolamine (PE), similar to the K+ activation of dephosphorylation in the sodium pump.

PE and PS are 10x as large as the cations transported by the sodium pump. This is known as the giant substrate problem. This work shows that isoleucine #364 (mutated in — patients with the ataxia, retardation and dysequilibrium syndrome Eur. J. Hum. Genet. vol. 21 pp. 281 – 285 ’13 aka CAMRQ syndrome ) forms a hydrophobic gate separating the entry and exit sites of PS. I364 likely directs the sequential formation and annihilation of water filled cavities (as shown by molecular dynamics simulations) allowing transport of the hydrophilic phospholipid head group, in a groove outlined by TMs 1, 2, 4 and 6, with the hydrocarbon chains following passively, still in the membrane lipid phase (and presumably outside the channel) — this must disrupt the hell out of the protein as it passes. They call this the credit card model — only the interaction with part of the molecule is important — just as the magnetic stripe is the only important thing about the credit card.

Are you sure you know everything your protein is up to?

Just because you know one function of a protein doesn’t mean you know them all. A recent excellent review of the (drumroll) executioner caspases [ Neuron vol. 88 pp. 461 – 474 ’15 ] brings this to mind. Caspases control a form of cell death called apoptosis, in which a cell goes gently into the good night without causing a fuss (particularly inflammation and alerting the immune system that something bad killed it). They are enzymes which chop up other proteins and cause the activation of other proteins which chop up DNA. They cause the inner leaflet of the plasma membrane to expose itself (particularly phosphatidyl serine which tells nearby scavenger cells to ‘eat me’).

The answer to the mathematical puzzle in the previous post will be found at the end of this one.

In addition to containing an excellent review of the various steps turning caspases on and off, the review talks about all the things activated caspases do in the nervous system without killing the neuron containing them. Among them are neurite outgrowth and regeneration of peripheral nerve axons after transection. Well that’s pathology, but one executioner caspase (caspase3) is involved in the millisecond to millisecond functioning of the nervous system — e.g. long term depression of neurons (LTD), something quite important to learning.

Of course, such potentially lethal activity must be under tight control, and there are 8 inhibitors of apoptosis (IAPs) of which 3 bind the executioners. We also have inhibitors of IAPs (SMAC, HTRA2) — wheels within wheels.

Are there any other examples where a protein discovered by one of its functions turns out to have others. Absolutely. One example is cytochrome c, which was found as it shuttles electrons to complex IVin the electron transport chain of mitochondria.Certainly a crucial function. However, when the mitochondria stops functioning either because it is told to or something bad happens, cytochrome c is released from mitochondria into the cytoplasm where it then activates caspase3, one of the executioner caspases.

Here’s another. Enzymes which hook amino acids onto tRNA are called tRNA synthases (aaRs for some reason). However one of the (called EPRS) when phosphorylated due to interferon gamma activity, became part of a complex of proteins which silences specific genes (translation — stops the gene from being transcribed) involved in the inflammatory response.

Yet another tRNA synthase, when released from the cell triggers an inflammatory response.

Naturally molecular biologists have invented a fancy word for the process of evolving a completely different function for a molecule — exaptation (to contrast it with adaptation).

Note the word molecule — exaptation isn’t confined to proteins. [ Cell vol. 160 pp. 554 – 566 ’15 ] Discusses exaptation as something which happens to promoters and enhancers. This work looked at the promoters and enhancers active in the liver in 20 mammalian species — all the enhancers were rapidly evolving.


Answer to the mathematical puzzle of the previous post. R is the set of 4 straight lines bounding a square centered at (0,0)

Here’s why proving it has an inside and an outside isn’t enough to prove the Jordan Curve Theorem

No. The argument for R uses its geometry (the boundary is made of straight
line segments). The problem is that an embedding f: S^1 -> R^2 may be
convoluted, say something of the the Hilbert curve sort.

Further (physical) chemical elegance

If the chemical name phosphatidyl serine (PS) draws a blank, read the verbatim copy of a previous post under the *** to find out why it is so important to our existence. It is an ‘eat me’ signal when there is lots of it around, telling professional scavenger cells to engulf the cell showing lots of PS on its surface.

Life, as usual, is more complicated. There are a variety of proteins exposed on cell surfaces which bind to phosphoserine. Not only that, but exposing just a little PS on the surface of a cell can trigger a protective immune response. Immune cells binding to just a little PS on the surface of another cell proliferate rather than eat the cell expressing the PS. This brings us to Proc. Natl. Acad. Sci. vol. 111 pp 5526 – 5531 ’14 that explains how a given PS receptor (called TIM4) acts differently depending how much PS is present.

Some PS receptors such as Annexin V have essentially an all or none response to PS, if they bind at all, they trigger a response in the cell carrying them. Not so for TIM4 which only reacts if there is a lot of PS around, leaving cells which express less PS alone. This allows these cells to function in the protective immune response.

So how does TIM4 do this? See if you can think of a mechanism before reading the rest.

In addition to the PS binding pocket TIM4 has 4 peripheral basic residues in separate places. The basic residues are positively charged at physiologic pH and bind to the negatively charged phosphate group of phosphatidyl serene or to the carboxylate anion of phosphatidyl serine. The paper doesn’t explain how these basic residues don’t bind to the other phospholipids of the cell surface (such as phosphatidyl choline or sphingomyelin). It is conceivable that the basic side chains (arginine, lysine etc.) are so set up that they only bind to carboxylate anions and not phosphate anions (but this is a stretch). That would at least give them specificity for phosphatidyl serene as opposed the other phospholipids present in both leaflets of the cell membrane. In any even TIM4 will be triggered only if these groups also bind PS, leaving cells which show relatively little PS alone. Clever no?

For the cognoscenti, the Hill coefficient of TIM4 is 2 while that of Annexin V is 8 (describing more than explaining the all or none character of Annexin V binding).

Flippase. Eat me signals. Dragging their tails behind them. Have cellular biologists and structural biochemists gone over to the dark side? It’s all quite innocuous as the old nursery rhyme will show

Little Bo Peep has lost her sheep
and doesn’t know where to find them
Leave them alone, and they’ll come home
wagging their tails behind them.

First, some cellular biochemistry. The lipid bilayer encasing all our cells is made of two leaflets, inner and outer. The composition of the two is different (unlike the soap bubble). On the inside we find phosphatidylethanolamine (PE), phosphatidylserine (PS). The outer leaflet contains phosphatidylcholine (PC) and sphingomyelin (SM) and almost no PE or PS. This is clearly a low entropy situation compared to having all 4 randomly dispersed between the 2 leaflets.

What is the possible use of this (notice how teleology invariably creeps into cellular biology)? Chemistry is powerless to explain such things. Much as I love chemistry, such truths must be faced.

It takes energy to maintain this peculiar distribution. The enzyme moving PE and PS back inside the cell is the flippase. It requires energy in the form of ATP to operate. When a cell is dying ATP drops, and entropy takes its course moving PE and PS to the cell surface. Specialized cells (macrophages) exist to scoop up the dying or dead cells, without causing inflammation. They recognize PE and PS by a variety of receptors and munch up cells exposing them on the surface. So PE and PS are eat me signals which appear when there isn’t enough ATP around for flippase to use to haul PE and PS back inside. Clever no?

No for some juicy chemistry (assuming that you consider transport of a molecule across a lipid bilayer actual chemistry — no covalent bonds to the transferred molecule are formed or removed, although they are to the transporter). Well it certainly is physical chemistry isn’t it?

Here are the structures of PE, PS, PC, SM http://www.google.com/search?q=phosphatidylserine&client=safari&rls=en&tbm=isch&tbo=u&source=univ&sa=X&ei=bDRLU5yfHOPLsQSOnoG4BA&ved=0CPABEIke&biw=1540&bih=887#facrc=_&imgdii=_&imgrc=qrLByG2vmhWdwM%253A%3BwAtgsTPwCxeZXM%3Bhttp%253A%252F%252Fscience.csumb.edu%252F~hkibak%252F241_web%252Fimg%252Fpng%252FCommon_Phospholipids.png%3Bhttp%253A%252F%252Fscience.csumb.edu%252F~hkibak%252F241_web%252Fcoursework_pages%252F2012_02_2.html%3B1297%3B934.

There are a few things to notice. Like just about every lipid found in our membranes, they are amphipathic — they have a very lipid soluble part (look at the long hydrocarbon changes hanging below them) and a very water soluble part — the head groups containing the phosphate.

This brings us to [ Proc. Natl. Acad. Sci. vol. 111 pp. E1334 – E1343 ’14 ] Which describes ATP8A2 (aka the flippase). Interestingly, the protein, with at least 10 alpha helices spanning the membrane, and 3 cytoplasmic domains closely resembles the classic sodium pump beloved of neurophysioloogists everywhere, which pumps sodium ions out of neurons and pumps potassium ions inside, producing the equally beloved membrane potential of neurons.

Look at those structures again. While there are charges on PE, PS (on the phosphate group), these molecules are far larger than the sodium or the potassium ion (easily by a factor of 10). This has long been recognized and is called the ‘giant substrate problem’.

The paper solved the structure of ATP8A2 and used molecular dynamics stimulations to try to understand how it works. What they found is that transmembrane alpha helices 1, 2, 4 and 6 (out of 10) form a water filled cavity, which dissolves the negatively charged phosphate of the head group. What happens to those long hydrocarbon tails? The are left outside the helices in the lipid core of the membrane. It is the charged head groups that are dragged through by the flippase, with the tails wagging along behind them, just like little Bo Peep.

There’s a lot more great chemistry in the paper, particularly how Isoleucine #364 directs the sequential formation and annihilation of the water filled cavities between alpha helices 1, 2, 4 and 6, and how a particular aspartic acid is phosphorylated (by ATP, explaining why the enzyme no longer works in energetically dying cells) changing conformation of all 10 transmembrane helices, so that only one half of the channel is open at a time (either to the inside or the outside).

Go read and enjoy. It’s sad that people who don’t know organic chemistry are cut off from appreciating such elegance. There is more to esthetics than esthetics.