Determinants of Unfolding Cooperativity and Binding Are Decoupled in a DNA Binding Domain

The relative magnitudes of noncovalent stabilization energies or the coupling free energies in folded proteins are anisotropically distributed, uniquely influencing folding and functional behaviors. In this regard, the fructose repressor (FruR) DBD belonging to the LacR repressor family harbors a three-residue insertion-KQY-between the canonical second and third helices. This sequence insertion promotes a strong Tyr-Tyr stacking interaction that is not observed in related homologues. Combining experiments with simulations, we show that the Tyr-Tyr stacking contributes to a decoupled unfolding due to the localization of a large part of the stabilization energy in this specific structural region. This leads to melting temperatures from different probes spanning nearly 10 K, while concomitantly stabilizing a partially structured intermediate state. Disruption of the aromatic stacking interaction via an alanine mutation promotes a molten-globular state whose native ensemble is replete with non-native interactions while displaying enhanced thermodynamic fluctuations and minimal calorimetric cooperativity. Surprisingly, the molten-globular variant of FruR DBD binds to the operator site on DNA with an affinity similar to that of the wild-type but with altered secondary-structure characteristics in the bound state, underscoring the chaperone-like role of DNA through its large negative electrostatic potential. FruR DBD thus appears to be at the verge of disorder as expected of an entropically destabilizing three-residue insertion but is rescued by the aromatic stacking interaction that distinctly dictates the finer details of stability, cooperativity, and binding.