Skip to Content

Biological News

Alarmingly Functional Disorder

Let’s think for a bit about how proteins bind to each other. After all, messing around with that is what keeps everyone in the drug industry employed, and the unmessed varieties of such binding events are what keep us all vertical and above room temperature, so it’s a worthy subject.

The mental picture is of two proteins adopting complementary shapes along some kinds of binding surfaces. “Complementary” is doing a lot of work in that sentence, though, because we could be talking about hydrophobic interactions (whatever those are), hydrogen bonding, or outright charged residues that are pairing up positive/negative. In fact, since we’re talking about proteins here, all of these can be operating at the same time, and probably are. And there are all sorts of entropic/enthalpic things going on, too – things are happening to water molecules at the surface of the proteins as they come together (as well as to the bulk water that used to be between them), parts of each protein are probably moving around and getting more or less constrained, other internal interactions within each partner are adjusting, etc. It’s a mess.

But it’s still a somewhat orderly mess. In the end, these two complex three-dimensional shapes have found some sort of defined relationship that’s overall lower-energy than what they started with, and now this is their new shape. What if that’s not the case, though? This unnerving thought is brought on by this paper, published late last month in Nature. We’re talking intrinsically disordered proteins again – those beasts, rather more common than was once thought, that have large sections of them that have no particular defined shape at all. (Indeed, some of them are disordered from snout to tail). I’ve generally thought of these, though, as flopping around in that way until they encounter a binding partner, at which point they settle down into some defined shape and slot themselves obligingly into my weltanschauung.

As should have been quite clear by now, though, proteins don’t care what I think about them. This paper shows a particular protein interaction (between histone H1 and prothymosin-alpha) that is down in the picomolar affinity range. The histone protein has a small structured region in the middle, but the N- and C-terminals head off into complete disorder. Prothymosin is disordered all the way through. If you’d asked me about this at one time, I would have been certain that this sort of binding required the formation of a solid, well-defined structure with plenty of clear interactions. But that’s not what’s going on. The paper shows that both proteins are still disordered even as a complex. In the NMR, you can see the structured globular part of the histone, but that’s the only order in sight. The circular dichroism spectra reflect this as well – the complex, in fact, is just the CD spectra of the two partners added on top of each other, with no sign of induced helicity, etc. The team did a whole series of FRET experiments, attaching the partner groups on a number of different residues, and there’s really no pattern to it at all.

What’s the interaction? Sheer charge. The partners are strongly positive/strongly negative, but they don’t seem to care what residues associate with what. Doubling the ionic strength of the buffer decreases the binding constant by six orders of magnitude, so yeah, it’s pretty much an ionic thing all the way. They’re just shifting around unfolded on a 100ns-timescale, with no apparent need for anything more organized.

There were already signs that something like this was going on. Such histone protein tails had already been shown, most disconcertingly, to bind their protein partners even when their sequences had been scrambled, and they’re not the only proteins that have demonstrated such behavior. It makes sense: if you don’t got no defined structure, you don’t need no defined sequence, right? Here’s a good try at classifying protein binding along scales of static/dynamic and order/disorder, with this latest example falling thoroughly into the “dynamic disordered” quadrant.

How should we think about this stuff? Well, for now, I’m modeling this in my head as “proteins have all sorts of binding modes that fit different needs in the cell”. There are some, obviously, that need pretty hard, defined structures both at their interface and in the other parts of the protein. There are some where a protein’s ordered regions bind other ordered regions, with disordered parts still boogieing around, and others where a totally disordered protein folds one end of its structure up into an ordered complex while still leaving the far end loose. All of that I’ve been able to handle without much problem. But, as this latest paper shows, we have to stretch this concept to include “disordered binding” itself. In these cases, I suppose that the key event is just bringing these proteins together somehow, without so much need for three-dimensional perfection.

The thing is, it looks like these sorts of disordered binding modes may be a lot more common in the proteome than any of us thought. We’re all going to have to accommodate that reality, apparently. Are we going to be able to attack such things with small molecules, though? I have to say that given the choice, I would try something else first. With a disordered protein we’ve always been able to make the argument that binding something to it in its unstructured state might throw its conformational manifold off enough to disturb its function – and of course, targeting it in a structured binding complex is always theoretically possible right up front. But picomolar-level binding that has no defined structure at the binding interface? I will read about this with interest, and I will think about its implications, but I would not like to lead a drug discovery project against it.

25 comments on “Alarmingly Functional Disorder”

  1. Marcin says:

    Happy Nowruz!

    1. Derek Lowe says:

      Eid-e shoma mubarak to you, too!

  2. luysii says:

    These unstructured proteins still have a structure amazingly enough. 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 (+ 53 or – 43 ). 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.

    For more on this see https://luysii.wordpress.com/2018/03/04/the-structure-of-an-unstructured-protein/

  3. Barry says:

    We’ve seen evidence for such non-ordered interactions in the cationic antibiotics e.g. polymyxins. They’re effective “small-molecule” drugs but the structural details don’t matter as much as the gross charge.

  4. Some idiot says:

    How about this? Let’s say that some proteins have a business end which need to bind to another protein for a while, but not too strongly, otherwise it will gum up the system. But the overall binding needs to be decent.

    So instead of trying to finely engineer something with tricky properties, just put an outrageously charged tail on it. That way, it will just mosey on around, but when it happens to be close enough to something of interest, the charged tail has taken away enough entropy so that the business end can get on with the work. Until the charged end finds something else to sniff at…

    I.e., cooperativity…

    1. Barry says:

      I presume something like that happens with most DNA-binding proteins. Charge-pair to associate with the phosphate backbone, something much more directional (or an RNA prosthesis?) to make the high-fidelity match

  5. Kelvin says:

    Ultimately, binding is no more than a statistical concept, where partners are more likely to be associated than not. And apparently, that requires no specific defined ordered structures to be formed at all, in some cases.

  6. a says:

    Is this going to be limited to something that has a huge # of charges – something tht has evolved to bind some massive poly-poly-polyanion like DNA?

  7. milkshake says:

    another example of very tight ionic binding between macromolecular targets that is poorly structured is heparin + protamin (=it is tight enough to be used as a heparin antidote)

  8. Yvar says:

    When looking for a model system for 1:1 protein interactions, our group came to the conclusion that few proteins in a cell interact in a pure binary fashion without any other proteins (or other more complex biologic polymers being involved). This really hurts my ability to understand how drugs affecting protein-protein interactions in cells really work with all those other dynamic complex members being involved.

    As an aside, beta-lactamase and beta-lactamase inhibitory protein (BLIP) was a system we found that did seem to truly be 1:1, at least in relevant bacteria.

    1. tangent says:

      So they mostly all have allosteric third parties at a minimum, or even less binary-ish relationships? That’s terrifying. How did you conclude that?

      1. Yvar says:

        Basically whenever we looked at a model protein and what it interacted with, nearly every protein had more than one interacting partner and its relationship couldn’t be described with simple 1:1 stoichiometry. I wouldn’t say it was a true conclusion, more an observation that further changed how complicated the cell seems to be.

  9. luysii says:

    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?

    1. DCRogers says:

      CDF(N=111, X=44, p=0.1) = 1.87 * 10^-16

      1. Passerby says:

        Luysii: “They are incredibly improbable on a purely statistical basis”

        And that’s why they have evolved piecewise. You are not making this argument yourself, but the ID crowd makes similar arguments about the improbability of molecular structures arising “by chance”. The fact of the matter is that simpler similar structures that were far more probable arose first and then were selected for by natural selection.

        1. David Edwards says:

          Not only that, but a number of the simpler antecedents selected for in the past, acquired new functions over time.

          Indeed, the whole “irreducible complexity” drivel spouted by the ID crowd, was destroyed by Hermann Joseph Müller back in 1918, over three decades before Behe was born, and six decades before he peddled his nonsense on the subject. Müller’s account of this can be found in the following paper:

          Genetic Variability, Twin Hybrids and Constant hybrids in a Case of Balanced Lethal Factors, by Hermann Joseph Müller, Genetics, 3(5): 422-499 (1918)

          The original paper can be downloaded for free here:

          Genetics Reprint Of 1918 Paper

          The relevant words were contained in pages 465-466, viz:

          Most present-day animals are the result of a long process of evolution, in which at least thousands of mutations must have taken place. Each new mutant in turn must have derived its survival value from the effect upon which it produced upon the ‘reaction system’ that had been brought into being by the many previously formed factors in cooperation; thus, a complicated machine was gradually built up whose effective working was dependent upon the interlocking action of very numerous different elementary parts or factors, and many of the characters and factors which, when new, were originally merely an asset finally became necessary because other necessary characters and factors had subsequently become changed so as to be dependent upon the former. It must result, in consequence, that a dropping out of, or even a slight change in any one of these parts is very likely to disturb fatally the whole machinery.

          In short, the Müllerian Two Step can be described thus:

          [1] Add a component;

          [2] Make it necessary.

          In short, Müller postulated that “irreducible complexity” was an outcome of evolutionary processes, not a refutation thereof. If Behe had done his homework, he would have known this.

          He’s since been given a thorough toasting over his assertions about the bacterial flagellum. See: Dover Trial. 🙂

  10. Ques says:

    The authors used ionic strength 200 mM or above. Is this common intracellular ionic strength?

    1. Katrine Bugge says:

      Hi Ques,
      No, besides the salt titration, the experiments where done at the physiologically relevant ionic strength of 165 mM.
      Cheers,
      Katrine

      1. Alessandro says:

        Thanks, Katrine!

  11. Jb says:

    2 proteins????? Lulz. Labmate right now is purifying 19-22 protein containing complexes.

    1. tlp says:

      do you mean ribosomes?

  12. Jb says:

    Also, intrinsically disordered domains ARE what governs life – maaaaaaaanny a transcription factor contain them.

  13. gippgig says:

    So does any strongly negative protein strongly bind any strongly positive protein? If not, why? How can there be any specificity?

  14. Foodscientist says:

    So they adopt the orgy motif?

  15. zero says:

    Structured proteins are dinner partners. They sit politely in their chairs and talk to their neighbors. Maybe their arms move around a bit, but the rest of them forms a defined shape.

    Disordered proteins are dance partners. They move and twist near each other while traveling around the dance floor interacting with all the other dancers.

Leave a Reply

Your email address will not be published. Required fields are marked *

Time limit is exhausted. Please reload CAPTCHA.