Allostery in the dynamic coactivator domain KIX occurs through minor conformational micro-states

Author summaryMost proteins involved in transcriptional regulation exhibit high degrees of flexibility, suggesting that mobility is integral to molecular recognition in such systems. Thus, establishing the molecular recognition principles could inform chemical probe development for selective control of gene expression. We interrogate the role of conformational entropy in the coactivator domain KIX of CBP in complex with different partners using simulations and fluorescence-based assays to show how the flexible domain engages with various partners. We find that the wide distribution of conformational micro-states in the apo ensemble facilitates recognition of multiple partners, where each activator preferentially binds to particular conformational states and in turn reweights the distribution of accessible micro-states. The coactivator KIX of CBP uses two binding surfaces to recognize multiple activators and exhibits allostery in ternary complex formation. Activator center dot coactivator interactions are central to transcriptional regulation, yet the microscopic origins of allostery in dynamic proteins like KIX are largely unknown. Here, we investigate the molecular recognition and allosteric manifestations involved in two KIX ternary systems c-Myb center dot KIX center dot MLL and pKID center dot KIX center dot MLL. Exploring the hypothesis that binary complex formation prepays an entropic cost for positive cooperativity, we utilize molecular dynamics simulations, side chain methyl order parameters, and differential scanning fluorimetry (DSF) to explore conformational entropy changes in KIX. The protein's configurational micro-states from structural clustering highlight the utility of protein plasticity in molecular recognition and allostery. We find that apo KIX occupies a wide distribution of lowly-populated configurational states. Each binding partner has its own suite of KIX states that it selects, building a model of molecular recognition fingerprints. Allostery is maximized with MLL pre-binding, which corresponds to the observation of a significant reduction in KIX micro-states observed when MLL binds. With all binding partners, the changes in KIX conformational entropy arise predominantly from changes in the most flexible loop. Likewise, we find that a small molecule and mutations allosterically inhibit/enhance activator binding by tuning loop dynamics, suggesting that loop-targeting chemical probes could be developed to alter KIX center dot activator interactions. Experimentally capturing KIX stabilization is challenging, particularly because of the disordered nature of particular activators. However, DSF melting curves allow for inference of relative entropic changes that occur across complexes, which we compare to our computed entropy changes using simulation methyl order parameters.