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Download fileStructural–Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble
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posted on 2022-01-27, 21:44 authored by Hemashree Golla, Adithi Kannan, Soundhararajan Gopi, Sowmiya Murugan, Lakshmi R Perumalsamy, Athi N. NaganathanThe 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.
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Keywords
transcriptional activity associatedtranscription factor tfiiordered protein domainsconformational selection mechanismprotein native ensemblenative ensemble characteristicsgoverning functional eventsdetermine fluctuation amplitudescompact native ensembleff1 domain samplesaltered structural couplingdamped equilibrium fluctuationsnative ensembleff1 domainturn determinefunctional outputequilibrium fluctuationswork demonstratesvitro underlying landscapethus establishsolvent exposureoften confoundedmultiple variablesmolecular simulationsmolecular basismodel systemk53 actingintimately tieddiverse arraycombining spectroscopyburied tyrosine