Allostery in the CBP KIX domain

Classically, allosteric and cooperative effects have been identified with large complexes of multiple protein subunits, in which the binding of a ligand to one subunit enhances ligand binding in a different subunit. While some features of the models developed to deal with these systems do not translate well to cases of allostery within a single protein or domain, many of their core ideas continue to illuminate these single-subunit systems. In an upcoming paper in the Journal of the American Chemical Society, a team of European researchers examine an allosteric effect based on population shifts in a transcriptional activator, comparing it to a famous model for explaining allostery in hemoglobin (1).

The CREB binding protein (CBP) is a large molecular scaffold that brings pieces of the transcriptional machinery together in order to turn on a gene. Like many scaffold proteins it contains several protein-protein interaction domains linked together by large unfolded regions. One of these domains is KIX, a small bundle of helices that binds other proteins at two distinct sites. In one case, a protein called MLL binds to one site while a protein called c-Myb binds at the other. What is so interesting about this is that KIX is much more likely to bind c-Myb when it is already bound to MLL. Brüschweiler et al. used NMR techniques to try and understand how this happens.

In order to pull this off they performed relaxation-dispersion experiments on the amide nitrogen, α-carbon, and some methyl carbon atoms of the KIX domain bound to a peptide derived from MLL. Many of the amino acids in the protein showed a significant contribution to R₂ from exchange, suggesting a global conformational switch between two states. In order to cover their bases, the authors performed experiments to prove that this behavior was not related to the unfolding of the protein. Satisfied that the protein was stable, they used standard methods to calculate the rate of the conformational change, the population of the two states, and the chemical shift difference between them. They found that the minor state of the KIX-MLL complex is 7% of the total population of protein molecules. They also noticed that the chemical shift difference between the two states correlates very well with the chemical shift difference between the KIX-MLL complex and the KIX-MLL-c-Myb complex. Assuming that the conformation of KIX is the primary determinant of chemical shift in the bound state, this suggests that the dynamics are sensing a switch between a state that doesn't bind c-Myb and a state that does.

In order to determine whether MLL binding gave rise to this conformational switching behavior, the authors measured relaxation dispersion in KIX at several different concentrations of MLL. Excluding residues highly sensitive (by chemical shift) to MLL binding, they found that the exchange contribution to relaxation increases as MLL is added. Although Brüschweiler et al. were unable to fit this small number of residues quantitatively, these results strongly suggest that the addition of MLL increases the population of the c-Myb binding state. Moreover, under conditions where KIX forms a saturated complex with MLL and a peptide from another protein (pKID), the chemical exchange contribution to relaxation vanishes, suggesting that the protein has been pushed completely to the binding-competent state.

In order to identify the pathway by which the MLL binding site communicates to the c-Myb binding site, the authors examined the residues in KIX that had the largest chemical shift change associated with the chemical exchange behavior. As it happens, residues satisfying these criteria cluster in a region stretching from the MLL site to the c-Myb site, as you can see to the right (explore this structure at the PDB). Here, KIX is blue, the MLL peptide is red, and the c-Myb peptide is green. The side chains of the residues Brüschweiler et al. identify are shown as sticks inside the pink atomic surface. As you can see, these residues constitute a contiguous body stretching from one site to the other. Presumably, this set of residues provides a pathway for communication between the two sites. A trio of isoleucines at the core of this region (I 611, 660, and 657) are present in KIX domains from many different species (supporting information), suggesting that this communication pathway is evolutionarily conserved. Mutational studies centered on this trio of residues may teach us more about the mechanism of information flow in this domain.

Although this allosteric pathway is known to work in reverse (binding of c-Myb enhances the binding of MLL), the authors were unable to detect any exchange contribution to R₂ when only c-Myb or pKID was bound. While this may suggest that communication in the opposite direction uses a completely different mechanism, such that KIX has two unidirectional allosteric pathways, that's not a necessary conclusion from this result. Alteration of R₂ due to conformational exchange is dependent on the populations of the two states, the difference in chemical shift between them, and the rate of the switch. Actually detecting a dispersion curve requires that all these parameters lie within a 'sweet spot' that allows observation. This doesn't always happen, even when a dynamic process is occurring with a μs-ms rate. Because the chemical shift changes that result from MLL binding appear to be quite large (2) the exchange process may be slow on the NMR timescale.

One minor concern I have with the paper is that the experiments were carried out at a pH of 5.8, which is lower than the pH of cytosol (7.2). The only groups likely to change their charge over that range are histidines, but one of the key residues for this paper is H651 in the KIX domain. The experiments that established the allosteric effect of MLL on c-Myb binding (2) were performed at pH 7.0 so it is formally possible that the dynamics and allostery are a coincidence (although the chemical shift data argue against this). It would probably be worthwhile to perform HMQC experiments to clarify the protonation state of the histidine, or to repeat the binding experiments at a lower pH. The latter might be preferable; I assume that mildly acidic conditions are used for the NMR experiments because KIX has undesirable spectral characteristics nearer neutral pH. Additionally, it might be interesting to perform experiments that explore the effects MLL has on the kinetics of binding, seeing as this appears to be a dynamic process.

Brüschweiler et al. identify their results with the Monod-Wyman-Changeux model of allostery. Although this model was formally developed for systems with multiple subunits, what the authors really wish to emphasize is the idea from the MWC model that proteins in solution exist in an equilibrium of high-affinity and low-affinity forms. The evidence from the relaxation-dispersion experiments indicates that a very small proportion of free KIX exists in a (unfavorable) conformation that's ready to bind c-Myb. The binding of MLL enhances KIX affinity for c-Myb by stabilizing this structure — the allosteric effect arises because MLL binding defrays the energetic cost of adopting this fold. This manifests as a shift in the population of KIX proteins towards the binding-competent state. This kind of binding cooperativity may play a significant role in CBP's transcriptional activation function.

(1) Sven Brüschweiler, Paul Schanda, Karin Kloiber, Bernhard Brutscher, Georg Kontaxis, Robert Konrat, Martin Tollinger (2009). Direct Observation of the Dynamic Process Underlying Allosteric Signal Transmission Journal of the American Chemical Society DOI: 10.1021/ja809947w

(2) N. K. Goto, T. Zor, M. Martinez-Yamout, H. J. Dyson, P. E. Wright (2002). Cooperativity in Transcription Factor Binding to the Coactivator CREB-binding Protein (CBP). Journal of Biological Chemistry, 277 (45), 43168-43174 DOI: 10.1074/jbc.M207660200