Tunable order–disorder continuum in protein–DNA interactions

Abstract

DNA-binding protein domains (DBDs) sample diverse conformations in equilibrium facilitating the search and recognition of specific sites on DNA over millions of energetically degenerate competing sites. We hypothesize that DBDs have co-evolved to sense and exploit the strong electric potential from the array of negatively charged phosphate groups on DNA. We test our hypothesis by employing the intrinsically disordered DBD of cytidine repressor (CytR) as a model system. CytR displays a graded increase in structure, stability and folding rate on increasing the osmolarity of the solution that mimics the non-specific screening by DNA phosphates. Electrostatic calculations and an Ising-like statistical mechanical model predict that CytR exhibits features of an electric potential sensor modulating its dimensions and landscape in a unique distance-dependent manner, while DNA plays the role of a non-specific macromolecular chaperone. Accordingly, CytR binds its natural half-site faster than the diffusion-controlled limit and even random DNA conforming to an electrostatic-steering binding mechanism. Our work unravels for the first time the synergistic features of a natural electrostatic potential sensor, a novel binding mechanism driven by electrostatic frustration and disorder, and the role of DNA in promoting distance-dependent protein structural transitions critical for switching between specific and non-specific DNA-binding modes.

Engineering Order and Cooperativity in a Disordered Protein

“…The CytR DM further binds to the udp half-site faster than the dead-time of the stopped flow instrument under pseudo first-order conditions (i.e. excess protein concentrations) setting the lower bound on k on for binding at >10 9 M -1 s -1 , similar to the WT48 (Figure S9).…”

“…CytR, PurR and their mutants were expressed and purified as described before 47, 48. All experiments were recorded in pH 7.0, 20 mM phosphate buffer (43 mM ionic strength) with or without urea.…”

“…Experimental conditions for CytR, PurR and their variants are identical unless otherwise mentioned. The details of dynamic light scattering experiments and electrostatic potential calculations can be found in references 47 and 48, respectively.…”

Post-translational modification analysis of Saccharomyces cerevisiae histone methylation enzymes reveals phosphorylation sites of regulatory potential

“…Sites on MTases were strongly enriched within disordered regions and at interaction interfaces but largely absent or under-represented on domains. This is functionally important as intrinsically disordered regions are known to mediate protein–protein interactions ( 74 ) and associations with nucleic acids ( 75 , 76 ). The positioning of phosphorylation observed here is also consistent with our previous study of phosphorylation on five non-histone protein MTases in yeast ( 39 ).…”

Dynamic Consequences of Specificity within the Cytidine Repressor DNA-Binding Domain

“…Thus, the CytR DBD can be characterized as an intrinsically disordered protein (IDP). Since our CytR DBD structure was published, it has been used as a paradigm in understanding IDPs in comparison to other folded LacR DBDs [7,[13][14][15][16][17][18]].…”