[MWClarkson blog] Dynamic origins of PBX1 homeodomain allostery

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Dynamic origins of PBX1 homeodomain allostery

In the Monod-Wyman-Changeux model for cooperative binding, proteins exist in an equilibrium of low-affinity and high-affinity states in solution, absent any ligand. In this view, although it may appear that the binding of a ligand causes a conformational transition, it actually stabilizes one conformation from a pre-existing equilibrium. In the past several years, advanced NMR techniques have yielded increasing evidence that these structural equilibria exist for a number of proteins, suggesting that this model for linkage between conformational change and binding may be quite general. An upcoming paper in the Journal of Molecular Biology (1) is typical of such findings.
Farber and Mittermaier studied the behavior of a homeodomain, a small, all-helical domain that typically binds to DNA, often in concert with other homeodomains. In particular, they were interested in the homeodomain of PBX1 (PBX-HD) which binds DNA cooperatively with a homeodomain from HOXB1 (HOX-HD). The domains interact with the DNA target and with each other. Peptides representing the binding site from the HOX-HD bind detectably to PBX-HD only in the presence of the target DNA, suggesting that the two binding sites communicate. The third helix of the PBX-HD is likely to mediate the allostery since it's involved in both binding interactions, but it's not clear from the available structures how this would happen. Additionally, there is a C-terminal sequence, with no defined fold in the free structure, that forms a helix in the ternary complex. It does not interact directly with the DNA, but removal of this extension decreases the affinity of PBX-HD for DNA and weakens the cooperativity between PBX-HD and HOX-HD.

Helix folding has a low energy barrier, so it is reasonable to suspect that this helix could form even in the absence of DNA. Farber and Mittermaier examined this possibility using a technique I have often discussed on this blog: CPMG relaxation dispersion. As you may recall, this technique is sensitive to fluctuations between states (chemical exchange) that persist for microseconds or milliseconds. One can in principle determine the rate of exchange (kex), the population of each state (pA, pB), and the chemical shift difference (|??|) between them, although if the motion is too fast or too slow only composites of some of these can be reliably determined. When they performed the experiment, the authors found that residues throughout PBX-HD had significant broadening, indicating chemical exchange and suggesting that the protein does not spend all its time in one folded state. The relaxation-dispersion profiles they obtained at 10 °C and 15 °C were in the intermediate regime, where all three of the aforementioned parameters can be determined.

For the C-terminal extension, the |??| determined by fitting the relaxation-dispersion data were linearly correlated with the chemical shift change that was observed in an HSQC upon binding (|??|). The correspondence wasn't exactly 1:1, but this is still reasonably good evidence that the helix is folding independent of binding. The authors used the |??| from the 10 °C fits to pull populations and rates from the experiments performed at higher temperatures, where only a composite parameter can be reliably determined (due to the speed of the fluctuation). Arrhenius plots derived from these data indicate thermodynamic parameters that are consistent with the folding of a single helix, again supporting the proposition that the C-terminal helix can fold on its own.

Numerous residues in the folded portion of the domain also experienced chemical exchange, which could mean that the helix is not the only thing undergoing a structural transition. The authors fit these residues individually, then tried again while fixing kex to the value determined from the helix behavior. The latter fits were not much worse in terms of their residuals than the floating fits were, so the fluctuations here could reasonably be seen as consistent with the helix-folding fluctuation.

If this is so, then removing this unstable helix should quench the dynamics in the folded part of the protein. This turned out to be the case — when the helix was removed, the dispersion curves for residues in the folded part of the protein became flat. This reinforces the case that the dynamics detected in the folded domain are related to the folding of the helix, and therefore represent an excursion to the "bound" structure for ligand-free protein.

Farber and Mittermaier note that for residues in the folded portion of the domain, the |??| determined through the CPMG analysis does not appear to agree with the |??| observed upon binding DNA. From this they conclude that the conformational change in solution is actually going to some unknown third state that is different from both the free and bound structures. I disagree somewhat with this interpretation. Because the ligand (in this case a piece of double-stranded DNA) is large relative to the protein and possesses substantial negative charge, there's a significant possibility of long-range electrostatic effects on the chemical shift of the PBX-HD. That is, the protein's bound state might have different chemical shifts free in solution and bound to the ligand even without any major conformational changes. If this is the case, the |??| will correlate best with |??| for residues that are far from the interface. Probably the structure sampled by the free protein is not exactly the same as the bound structure, but I think further data would be needed to determine whether the alternative structure in the free state differs significantly from the bound structure with DNA.

The uncertainty about the alternative structural state of the free protein makes it more difficult to make a firm argument about whether the binding mechanism more closely resembles conformational selection or induced fit, or whether it's some kind of middle ground between the two. Although it's suggestive, the observation of a structural equilibrium in the free state does not actually indicate how binding occurs. Moreover, because this is a complicated ternary complex, it is possible that, say, the protein-binding mechanism is conformational selection, while the DNA mechanism is induced-fit. This latter possibility might seem more sensible in light of existing studies indicating that long-range (e.g. electrostatic) interactions may predispose a system to induced-fit binding.

Complications aside, these data seem to support a model in which the PBX-HD transiently adopts the bound conformation in the absence of ligand. Binding of the PBX-HD domain to DNA shifts its population towards the state that is the minority in solution. This new structure has high affinity for the HOX-HD, promoting the formation of the ternary complex. In principle, binding of the HOX-HD to PBX-HD could precede DNA binding by both modules, but the interaction between these proteins appears to be weak in the absence of DNA. However, proving that the excursion to the bound (or near-bound) PBX-HD structure represents an actual intermediate in the binding process rather than just an interesting fluctuation on the side will require some determination of the binding kinetics in various conditions.

(1) Farber, P., & Mittermaier, A. (2010). Concerted Dynamics Link Allosteric Sites in the PBX Homeodomain Journal of Molecular Biology DOI: 10.1016/j.jmb.2010.11.016


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