Binding-Induced Folding of a Natively Unstructured Transcription Factor

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Abstract
Transcription factors are central components of the intracellular regulatory networks that control gene expression. An increasingly recognized phenomenon among human transcription factors is the formation of structure upon target binding. Here, we study the folding and binding of the pKID domain of CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based Gō-type model predict a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and Φ-value analyses, we find that the binding transition state resembles the unstructured state in solution, implying that CREB becomes structured only after committing to binding. A change of structure following binding is reminiscent of an induced-fit mechanism and contrasts with models in which binding occurs to pre-structured conformations that exist in the unbound state at equilibrium. Interestingly, increasing the amount of structure in the unbound pKID reduces the rate of binding, suggesting a “fly-casting”-like process. We find that the inclusion of attractive non-native interactions results in the formation of non-specific encounter complexes that enhance the on-rate of binding, but do not significantly change the binding mechanism. Our study helps explain how being unstructured can confer an advantage in protein target recognition. The simulations are in general agreement with the results of a recently reported nuclear magnetic resonance study, and aid in the interpretation of the experimental binding kinetics. Protein-protein interactions are central to many physiological processes. Traditionally, the atomic structure of the isolated proteins, as obtained from X-ray crystallography or NMR spectroscopy, has been thought to determine the molecular recognition and binding process. However, this view has been challenged by the discovery of natively unstructured proteins, including many human transcription factors, which assume ordered molecular structures only upon binding to their targets. Understanding these transitions from a dissociated and unfolded state to a bound and folded state is key to unraveling how transcription factors, proteins involved in the system that controls the transcription of genetic information from DNA to RNA, perform their function. We conducted molecular simulations to study the binding of a well characterized transcription/co-transcription factor complex. We found that the transcription factor is unstructured when binding to its partner, with folding into an ordered structure occurring only after the initial binding event. The resulting coupled folding-and-binding mechanism is found to be in accord with state-of-the-art experimental data. Transcription-factor binding in an unstructured state may confer an advantage in protein target recognition by accelerating the rate of association without compromising the ability to bind to a diverse set of proteins with high specificity and yet relatively low affinity.