Structural-Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble

The functioning of proteins is intimately tied to their fluctuations in the native ensemble. The structural-energetic features that determine fluctuation amplitudes and hence the shape of the underlying landscape, which in turn determine the magnitude of the functional output, are often confounded by multiple variables. Here, we employ the FF1 domain from human p190A RhoGAP protein as a model system to uncover the molecular basis for phosphorylation of a buried tyrosine, which is crucial to the transcriptional activity associated with transcription factor TFII-I. Combining spectroscopy, calorimetry, statistical- mechanical modeling, molecular simulations, and in vitro phosphorylation assays, we show that the FF1 domain samples a diverse array of conformations in its native ensemble, some of which are phosphorylation-competent. Upon eliminating unfavorable charge- charge interactions through a single charge-reversal (K53E) or charge-neutralizing (K53Q) mutation, we observe proportionately lower phosphorylation extents due to the altered structural coupling, damped equilibrium fluctuations, and a more compact native ensemble. We thus establish a conformational selection mechanism for phosphorylation in the FF1 domain with K53 acting as a gatekeeper, modulating the solvent exposure of the buried tyrosine. Our work demonstrates the role of unfavorable charge-charge interactions in governing functional events through the modulation of native ensemble characteristics, a feature that could be prevalent in ordered protein domains.

Automated summary

The fluctuations in protein's native ensemble play a significant role in its functioning and determining the functional output is confounded by multiple variables. Through the study of the FF1 domain in human p190A RhoGAP protein, it was discovered that phosphorylation of a buried tyrosine, which is crucial in transcriptional activity, is regulated through the modulation of structural coupling and native ensemble characteristics. The unfavorable charge-charge interactions were found to be important in governing this functional event.