Protein plasticity driven by disorder and collapse governs the heterogeneous binding of CytR to DNA

Abstract

The amplitude of thermodynamic fluctuations in biological macromolecules determines their conformational behavior, dimensions, nature of phase transitions and effectively their specificity and affinity, thus contributing to fine-tuned molecular recognition. Unique among large-scale conformational changes in proteins are temperature-induced collapse transitions in intrinsically disordered proteins (IDPs). Here, we show that CytR DNA-binding domain, an IDP that folds on binding DNA, undergoes a coil-to-globule transition with temperature in the absence of DNA while exhibiting energetically decoupled local and global structural rearrangements, and maximal thermodynamic fluctuations at the optimal bacterial growth temperature. The collapse is shown to be a continuous transition through a combination of statistical-mechanical modeling and all-atom implicit solvent simulations. Surprisingly, CytR binds single-site cognate DNA with negative cooperativity, described by Hill coefficients less than one, resulting in a graded binding response. We show that heterogeneity arising from varying binding-competent CytR conformations or orientations at the single-molecular level contributes to negative binding cooperativity at the level of bulk measurements due to the conflicting requirements of collapse transition, large fluctuations and folding-upon-binding. Our work reports strong evidence for functionally driven thermodynamic fluctuations in determining the extent of collapse and disorder with implications in protein search efficiency of target DNA sites and regulation.

Tunable order–disorder continuum in protein–DNA interactions

“…The fluorescence emission of tyrosine exhibits a small red-shift on increasing temperatures providing an alternate probe to monitor structural changes. This component of the spectra can be extracted by performing singular value decomposition of the global temperature–wavelength–ionic strength data (Figure 2C ) ( 36 ). Such an analysis reveals that the red-shift dominates the observed spectrum at ∼283 K in the presence of urea wherein the protein is fully unfolded, while it increases to ∼301 K at 43 mM ionic strength and eventually to just ∼319 K at 1.7 M ionic strength (Figure 2D ).…”

“…The protocol for overexpression of CytR, purification and spectroscopic measurements is outlined in detail in ( 36 ). The 2D [ 15 N, 1 H]-HSQC spectra were recorded at 298 K on a Bruker Avance III 800 MHz nuclear magnetic resonance (NMR) spectrometer equipped with a cryogenically cooled triple resonance probe.…”

“…The scanning calorimetry experiments were recorded in a MicroCal VP-Capillary DSC with an automated sample injector as described before ( 36 ). The variable-barrier (VB) model analysis was performed on the absolute heat capacity thermograms of CytR at the three explored ionic strength conditions by fixing the Freire folded baseline ( 49 ).…”

“…This can lead to several interesting folding–binding–translocation–regulation mechanisms—folding-rate acceleration ( 12 ), folding-upon-binding/binding-upon-folding ( 13 , 14 ) or combination of the two ( 15 ), synergistic folding ( 14 , 16 , 17 ), ‘fly-casting’ ( 18 , 19 ), ‘monkey-bar’ binding ( 20 ), conformational switching ( 21–26 ), homo- versus heterodimerization ( 27 ) and protein co-localization ( 28–30 )—all of which determine the extent of time the protein spends bound to DNA, its 1D diffusion coefficient relative to the time spent freely diffusing in solution ( 14 , 31–34 ) and hence fine-tuned expression of genes. In many cases, the differences between specific and non-specific binding poses are subtle ( 8 , 11 , 22 , 35 ) and can result in distinct cooperative effects ( 21 , 36 ).…”

“…Cytidine repressor (CytR) DBD (referred to as CytR) is an intrinsically disordered protein (IDP) that binds its target udp half-site with a weak affinity ( 46 ), and promiscuously to multiple other sites ( 47 ), despite assuming a folded-like structure on binding. In fact, a detailed analysis revealed that CytR is highly frustrated electrostatically, samples multiple conformations in equilibrium driven by non-specific and a continuous collapse transition, but this conformational heterogeneity is also translated into binding heterogeneity ( 36 ), reminiscent of the ‘fuzzy’ complexes in protein–protein interactions ( 48 ).…”