The cellular response to hypoxia is critical for cell survival and is fine-tuned to allow cells to recover from hypoxic stress and adapt to heterogeneous or fluctuating oxygen levels1,2. The hypoxic response is mediated by the α subunit of the transcription factor HIF-1 (HIF-1α)3, which interacts via its intrinsically disordered C-terminal transactivation domain with the TAZ1 (CH1) domain of the general transcriptional coactivators CBP and p300 to control transcription of critical adaptive genes4–6. One such gene is CITED2, a negative feedback regulator that attenuates HIF transcriptional activity by competing for TAZ1 binding through its own disordered transactivation domain7–9. Little is known about the molecular mechanism by which CITED2 displaces the tightly bound HIF-1α from their common cellular target. The HIF-1α and CITED2 transactivation domains bind to TAZ1 through helical motifs that flank a conserved LP(Q/E)L sequence that is essential for negative feedback regulation5,6,8,9. We show that CITED2 displaces HIF-1α by forming a transient ternary complex with TAZ1 and HIF-1α and competing for a shared binding site via its LPEL motif, thus promoting a conformational change in TAZ1 that increases the rate of HIF-1α dissociation. Through allosteric enhancement of HIF-1α release, CITED2 activates a highly responsive negative feedback circuit that rapidly and efficiently attenuates the hypoxic response, even at modest CITED2 concentrations. This hypersensitive regulatory switch is entirely dependent on the unique flexibility and binding properties of these intrinsically disordered proteins and exemplifies a likely common strategy used by the cell to respond rapidly to environmental signals.

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo or off-resonance R 1rho pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Deltaomega) between the conformations. The CPMG experiments were applied to the backbone N-H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme-substrate (ES) complex, and enzyme-product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s (-1) and is the same as both k cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k ex derived from the NMR measurements and k cat from enzyme kinetic studies. This isotope effect of approximately 2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function. For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R 1rho experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10,000 s (-1), which is the same as the catalytic rate constant. The O (eta) atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in ...

Intrinsically disordered proteins participate in many important cellular regulatory processes. The absence of a well-defined structure in the free state of a disordered domain, and even on occasion when it is bound to physiological partners, is fundamental to its function. Disordered domains are frequently the location of multiple sites for post-translational modification, the key element of metabolic control in the cell. When a disordered domain folds upon binding to a partner, the resulting complex buries a far greater surface area than in an interaction of comparably-sized folded proteins, thus maximizing specificity at modest protein size. Disorder also maintains accessibility of sites for post-translational modification. Because of their inherent plasticity, disordered domains frequently adopt entirely different structures when bound to different partners, increasing the repertoire of available interactions without the necessity for expression of many different proteins. This feature also adds to the faithfulness of cellular regulation, as the availability of a given disordered domain depends on competition between various partners relevant to different cellular processes.

Allosteric regulatory processes are implicated at all levels of biological function. Recent advances in our understanding of the diverse and functionally significant class of intrinsically disordered proteins have identified a multitude of ways in which disordered proteins function within the confines of the allosteric paradigm. Allostery within or mediated by intrinsically disordered proteins ensures robust and efficient signal integration through mechanisms that would be extremely unfavorable or even impossible for globular protein interaction partners. Here, we highlight recent examples that indicate the breadth of biological outcomes that can be achieved through allosteric regulation by intrinsically disordered proteins. Ongoing and future work in this rapidly evolving area of research will expand our appreciation of the central role of intrinsically disordered proteins in ensuring the fidelity and efficiency of cellular regulation.

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein (CBP) by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To obtain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used NMR spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility of HIF-1α observed in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α and CITED2 bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.