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.