The uses of disorder in the cell

We know that many proteins have disordered segments, and an older (2004) estimate says that over 30% of all eukaryotic proteins have disordered stretches of more than 30 amino acids.  Here is another example where the disordered conformation(s) of a protein is the form used by the cell.

Histone H1 (aka the linker histone) binds to DNA between nucleosomes.  It is thought to be important in the 10,000 or so compaction of the 3 meters or so of DNA each cell has so it fits into a 10 micron nucleus.  Histone H1 has a disordered carboxy terminal tail of 100 amino acids.  Unsurprisingly it is strongly positively charged (so it binds to the negatively charged phosphates holding DNA together).

H1 was studied in an interesting paper [ Proc. Natl. Acad. Sci. vol. 115 pp. 11964 – 11969 ’18 ].  The tail was added to short (36 basepairs) double stranded segment of DNA, under various stoichiometries and ionic compositions.  They found regions where the complex formed liquid droplets the size of microns.

We know DNA is compacted and people have looked for the 30 nanoMeter DNA fiber of DNA bound to nucleosomes for years without success.  It is possible that the compaction in DNA is due to phase separation (which is basically unstructured) rather than the rather specific structures proposed.  H1 may be acting as a likquidlike glue.  Fascinating.

In other work H1 was complexed with another protein (Prothymosin alpha) which is another intrinsically disordered protein which actually serves as a histone H1 chaperone.  Prothymosin is is polyAnionic, so it binds to polyCationic H1.  What is fascinating is that the binding is quite tight (picoMolar) and yet even when so tightly bound H1 remains disordered, something to confound drug chemists who are always looking for specific binding conformations.

The paper also describes Psi DNA, which is formed in solutions of cationic polymers. Here DNA condenses into a compact solvent excluded state.  It is an ordered assembly of B-DNA arranged in parallel twisted helical segments with a well define spacing.  It produces an anomalously large scattering signal in circular dichroism spectra.

Here is an older post in which the functional form of a protein is the unstructured one

When the active form of a protein is intrinsically disordered

Back in the day, biochemists talked about the shape of a protein, influenced by the spectacular pictures produced by Xray crystallography. Now, of course, we know that a protein has multiple conformations in the cell. I still find it miraculous that the proteins making us up have only relatively few. For details see — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/.

Presently, we also know that many proteins contain segments which are intrinsically disordered (e.g. no single shape).The pendulum has swung the other way — “estimations that contiguous regions longer than 50 amino acids ‘may be present” in ‘up to’ 50% of proteins coded in eukaryotic genomes [ Proc. Natl. Acad. Sci. vol. 102 pp. 17002 – 17007 ’05 ]

[ Science vol. 325 pp. 1635 – 1636 ’09 ] Compared to ordered regions, disordered regions of proteins have evolved rapidly, contain many short linear motifs that mediate protein/protein interactions, and have numerous phosphorylation sites compared to ordered regions. Disordered regions are enriched in serine and threonine residues, while ordered sequences are enriched in tyrosines — this highlights functional differences in the types of phosphorylation. Interestingly tyrosines have been lost during evolution.

What are unstructured protein segments good for? One theory is that the disordered segment can adopt different conformations to bind to different partners — this is the moonlighting effect. Then there is the fly casting mechanism — by being disordered (hence extended rather than compact) such proteins can flail about and find partners more easily.

Given what we know about enzyme function (and by inference protein function), it is logical to assume that the structured form of a protein which can be unstructured is the functional form.

Not so according to this recent example [ Nature vol. 519 pp. 106 – 109 ’15 ]. 4EBP2 is a protein involved in the control of protein synthesis. It binds to another protein also involved in synthesis (eIF4E) to suppress a form of translation of mRNA into protein (cap dependent translation if you must know). 4EBP2 is intrinsically disordered. When it binds to its target it undergoes a disorder to ordered transition. However eIF4E binding only occurs from the intrinsically disordered form.

Control of 4EBP2 activity is due, in part, to phosphorylation on multiple sites. This induces folding of amino acids #18 – #62 into a 4 stranded beta domain which sequesters the canonical YXXXLphi motif with which 4EBP2 binds eIF4E (Y stands for tyrosine, X for any amino acid, L for leucine and phi for any bulky hydrophobic amino acid). So here we have an inactive (e.g. nonbonding) form of a protein being the structured rather than the unstructured form. The unstructured form of 4EBP2 is therefore the physiologically active form of the protein.

Homework assignment and answer

A few days ago I gave the following homework assignment for the ace protein chemist, and promised an answer.

Here’s the assignment

Homework assignment for the protein chemist

As an ace protein chemist you are asked to design two proteins, both intrinsically disordered which form a tight complex with a picoMolar dissociation constant.  To make the problem ‘easier’ there is no need for specific amino acid interactions between the proteins.  To make the problem harder, even in the tight complex formed, the two proteins remain intrinsically disordered.

Hint: ‘nature’, ‘evolution’, ‘God’ —  whatever you chose to call it, has solved the problem.

Here’s the answer

The structure of an unstructured protein

Protein structure without structure.  No I haven’t fallen under the spell of a Zen master.  As Bill Clinton would say, it depends on what you mean by structure.

If you mean a segment of the protein chain which doesn’t settle down into one structure, you are talking about intrinsically disordered proteins. It is estimated that 40% of all human proteins contain at least one intrinsically disordered segment of 30 amino acids or more ( Nature vol. 471 pp. 151 – 153 ’11 ).   The same paper ‘estimates’ that 25% of all human proteins are likely to be disordered from beginning to end.

Frankly, I’ve always been amazed that any protein settles down into one shape — for details please see — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/. But that’s ‘old news’ as another Clinton would say.

Two fascinating papers in the current Nature (vol. 555 pp. 37 – 38, 61 – 66 ’18 1 March) describe the interaction of two very unstructured proteins.  One is prothymosin-alpha with 111 amino acids and a net negative charge of -44.  The other is Histone H1 with at least 189 amino acids and a net positive charge of + 53.  With such a charge imbalance it’s unlikely that they can coalesce into a compact single form.  So they are both intrinsically disordered proteins.

However the two proteins bind to each other quite tightly (dissociation constant is in the picoMolar range).  Even when they form a complex, a variety of techniques (NMR, single molecule fluorescent techniques, computation) show that neither settles down into a single form and are still unstructured.

So where’s the structure?  It isn’t in the amino acid sequence.  It isn’t the conformations adopted in space.  The structure is  in the net charge.  Many intrinsically disordered proteins have levels of net charge similar to those of prothymosin alpha and histone H1.  In the human proteome alone, several hundred proteins that are predicted to be intrinsically disordered contain contiguous stretches of at least 50 residues with a fractional net charge similar to that of H1 or proThymosin alpha (Bioinformatics 21, 3433–3434 2005) — hopefully there’s something newer.

The amino-acid sequences of disordered regions in proteins evolve rapidly, yet (Proc. Natl. Acad. Sci vol. 114 pp. E1450–E1459 2017) showed that the net charge is conserved despite a high degree of sequence diversity .  This should be a current enough reference.

Why in the world would the cell have something like this?  Most readers probably know what histones are.  If so, stop and think how the binding of the two proteins could be used by the cell before reading what the authors say about it.

“The interaction mechanism of proThymosin alpha and Histone H1 probably aids their biological function.  proThymosin alpha assists with the assembly and disassembly of chromatin, the material in which DNA is packaged with histone proteins (such as H1) in cells. To perform its function, proThymosin alpha must recognize its histone substrates rapidly and with sufficient affinity to compete with the high affinity of histone–DNA interactions (a similar high positive charge high negative charge interaction). The high binding affinity of Pro-Tα for H1 and the association rate of the two pro-teins imply that the dissociation of proThymosin alpha–H1 complexes is slow enough to allow functional outcomes, but fast enough not to slow down biological turnover.”

Why don’t they form a coacervate — a bunch of molecules held together by hydrophobic forces? Why don’t they show liquid liquid phase separations? The authors speculate that it might be due to the complementarity of the two proteins in terms of effective length and opposite net charge. Also they don’t have hydrophobic and aromatic side chains and cation pi interations which are said to favor phase separation mediated by proteins.

Addendum 20 March’18 — from my comment and a response on Derek’s blog

Luysii:

Another way to look at these very charge imbalanced proteins, is that they are being strongly (and positively) selected for. They are incredibly improbable on a purely statistical basis. Prothymosin alpha has 111 amino acids of which 44 are negatively charged. There are 20 amino acids of which only 2 (glutamic acid and aspartic acid) have negative charges at physiologic pH — cysteine and tyrosine can form anions but under much more basic conditions. So, assuming a random assortment of amino acids, the idea that 10% of the amino acids could fight for space with 90% of the rest and win around 40% of the time in 111 battles is extremely improbable. You’d have to use Stirling’s approximation for factorials to figure out exactly how improbable this is. Any takers?

Reply
DCRogers says:
March 20, 2018 at 2:35 pm
CDF(N=111, X=44, p=0.1) = 1.87 * 10^-16

The structure of an unstructured protein

Protein structure without structure.  No I haven’t fallen under the spell of a Zen master.  As Bill Clinton would say, it depends on what you mean by structure.

If you mean a segment of the protein chain which doesn’t settle down into one structure, you are talking about intrinsically disordered proteins. It is estimated that 40% of all human proteins contain at least one intrinsically disordered segment of 30 amino acids or more ( Nature vol. 471 pp. 151 – 153 ’11 ).   The same paper ‘estimates’ that 25% of all human proteins are likely to be disordered from beginning to end.

Frankly, I’ve always been amazed that any protein settles down into one shape — for details please see — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/. But that’s ‘old news’ as another Clinton would say.

Two fascinating papers in the current Nature (vol. 555 pp. 37 – 38, 61 – 66 ’18 1 March) describe the interaction of two very unstructured proteins.  One is prothymosin-alpha with 111 amino acids and a net negative charge of -44.  The other is Histone H1 with at least 189 amino acids and a net positive charge of + 53.  With such a charge imbalance it’s unlikely that they can coalesce into a compact single form.  So they are both intrinsically disordered proteins.

However the two proteins bind to each other quite tightly (dissociation constant is in the picoMolar range).  Even when they form a complex, a variety of techniques (NMR, single molecule fluorescent techniques, computation) show that neither settles down into a single form and are still unstructured.

So where’s the structure?  It isn’t in the amino acid sequence.  It isn’t the conformations adopted in space.  The structure is  in the net charge.  Many intrinsically disordered proteins have levels of net charge similar to those of prothymosin alpha and histone H1.  In the human proteome alone, several hundred proteins that are predicted to be intrinsically disordered contain contiguous stretches of at least 50 residues with a fractional net charge similar to that of H1 or proThymosin alpha (Bioinformatics 21, 3433–3434 2005) — hopefully there’s something newer.

The amino-acid sequences of disordered regions in proteins evolve rapidly, yet (Proc. Natl. Acad. Sci vol. 114 pp. E1450–E1459 2017) showed that the net charge is conserved despite a high degree of sequence diversity .  This should be a current enough reference.

Why in the world would the cell have something like this?  Most readers probably know what histones are.  If so, stop and think how the binding of the two proteins could be used by the cell before reading what the authors say about it.

“The interaction mechanism of proThymosin alpha and Histone H1 probably aids their biological function.  proThymosin alpha assists with the assembly and disassembly of chromatin, the material in which DNA is packaged with histone proteins (such as H1) in cells. To perform its function, proThymosin alpha must recognize its histone substrates rapidly and with sufficient affinity to compete with the high affinity of histone–DNA interactions (a similar high positive charge high negative charge interaction). The high binding affinity of Pro-Tα for H1 and the association rate of the two pro-teins imply that the dissociation of proThymosin alpha–H1 complexes is slow enough to allow functional outcomes, but fast enough not to slow down biological turnover.”

Why don’t they form a coacervate — a bunch of molecules held together by hydrophobic forces? Why don’t they show liquid liquid phase separations? The authors speculate that it might be due to the complementarity of the two proteins in terms of effective length and opposite net charge. Also they don’t have hydrophobic and aromatic side chains and cation pi interations which are said to favor phase separation mediated by proteins.

Short and Sweet

Yamanaka strikes again. Citrulline is deiminated arginine, replacing a C=N-H (the imine) by a carbonyl C=O. An enzyme called PAD4 does the job. Why is it important? Because one of its targets is the H1 histone which links nucleosomes together. Recall that the total length of DNA in each and every one of our cells is 3 METERS. By wrapping the double helix around nucleosomes, the DNA is shortened by one order of magnitude.

So what? Well, at physiologic pH the imine probably binds another proton making it positively charged, making it bind to the negatively charged DNA phosphate backbone. Removing the imine makes this less likely to happen, so the linker doesn’t bind the double helix as tightly.

Duck soup for the chemist, but apparently no one had thought to look at this before.

This opens up the DNA (aka chromatin decondensation) for protein transcription. Why is Yamanaka involved? Because PAD4 is induced during cellular reprogramming to induced pluripotent stem cells (iPSCs), activating the expression of key stem cell genes. Inhibition of PAD4 lowers the percentage of pluripotent stem cells, reducing reprogramming efficiency. The paper is Nature vol. 507 pp. 104 – 108 ’14.

Will this may be nice for forming iPSCs, it should be noted that PAD4 is unregulated in a variety of tumors.