Effect of osmolytes on the binding of EGR1 transcription factor to DNA

Abstract

Osmolytes play a key role in maintaining protein stability and mediating macromolecular interactions within the intracellular environment of the cell. Herein, we show that osmolytes such as glycerol, sucrose, and polyethylene glycol 400 (PEG400) mitigate the binding of early growth response (protein) 1 (EGR1) transcription factor to DNA in a differential manner. Thus, while physiological concentrations of glycerol only moderately reduce the binding affinity, addition of sucrose and PEG400 is concomitant with a loss in the binding affinity by an order of magnitude. This salient observation suggests that EGR1 is most likely subject to conformational equilibrium and that the osmolytes exert their effect via favorable interactions with the unliganded conformation. Consistent with this notion, our analysis reveals that while EGR1 displays rather high structural stability in complex with DNA, the unliganded conformation becomes significantly destabilized in solution. In particular, while liganded EGR1 adopts a well-defined arc-like architecture, the unliganded protein samples a comparatively large conformational space between two distinct states that periodically interconvert between an elongated rod-like shape and an arc-like conformation on a submicrosecond time scale. Consequently, the ability of osmolytes to favorably interact with the unliganded conformation so as to stabilize it could account for the negative effect of osmotic stress on EGR1-DNA interaction observed here. Taken together, our study sheds new light on the role of osmolytes in modulating a key protein-DNA interaction.

Keywords: conformational; equilibrium; molecular dynamics; osmotic stress; protein-DNA thermodynamics.

Figure 1

Electrostatic surface potential map derived from the structural model of the DB domain of EGR1 in complex with ZRE duplex. The relative location of three tandem zinc fingers (denoted ZFI, ZFII and ZFIII) is indicated on the composite molecular surface of the DB domain. Note that three different orientations of the DB domain, related by a 90°-clockwise rotation about the vertical axis in successive order from left to right, are shown for the inquisitive eye. The blue and red colors respectively denote the density of positive and negative charges, while the apolar and polar surfaces are indicated by white/gray color on the molecular surface. The ZRE duplex is displayed as a “stick” model and colored yellow.

Figure 2

Representative ITC isotherms for the binding of ZRE duplex to DB domain of EGR1 at sucrose concentrations of 0mM (a), 200mM (b) and 400mM (c). The upper panels show raw ITC data expressed as change in thermal power with respect to time over the period of titration. In the lower panels, change in molar heat is expressed as a function of molar ratio of ZRE duplex to DB domain. The red solid lines in the lower panels show the fit of data to a one-site binding model using the integrated NanoAnalyze software as described previously (31, 33).

Figure 3

Effect of varying concentrations of glycerol (a), sucrose (b) and PEG400 (c) on the apparent equilibrium dissociation constant (K_d) associated with the binding of ZRE duplex to DB domain of EGR1. In all panels, the red solid lines represent exponential fits to data points. The error bars were calculated from at least three independent measurements to one standard deviation.

Figure 4

Effect of increasing concentrations of glycerol (a), sucrose (b) and PEG400 (c) on enthalpic (ΔH°) and entropic (TΔS°) contributions to the free energy (ΔG°) accompanying the binding of ZRE duplex to DB domain of EGR1. In the top and middle panels, the solid lines are used to connect data points for clarity. In the bottom panels, the red solid lines represent linear fits to data points. The error bars were calculated from at least three independent measurements to one standard deviation.

Figure 5

Far-UV CD analysis of DB domain of EGR1 in the absence of osmolytes (black) and pre-equilibrated with 1000mM glycerol (red), 400mM sucrose (green), and 200mM PEG400 (blue). (a) Representative far-UV spectra at 25°C. (b) Representative melting curves over the temperature (T) range 20–100°C expressed in terms of the mean ellipticity observed at a wavelength of 222nm, [θ₂₂₂]. The solid lines through each data set represent non-linear least-squares fits to a two-state model using the ORIGIN software. The values determined for the melting transition (T_m) from these fits under each condition are indicated in the corresponding parenthesis.

Figure 6

Representative CD spectra of ZRE duplex in the absence of osmolytes (black) and pre-equilibrated with varying concentrations of glycerol (a), sucrose (b), and PEG400 (c) as indicated.

Figure 7

Structural stability of EGR1 as probed through MD simulations conducted on the structural models of DB domain bound to DNA (liganded) and in the absence of DNA (unliganded). (a) Root mean square deviation (RMSD) of backbone atoms (N, Cα and C) within each simulated structure relative to the initial modeled structure of liganded (top panel) and unliganded (bottom panel) protein as a function of simulation time. Note that the overall RMSD of the DB domain (black) is deconvoluted into its three constituent zinc fingers: ZFI (green), ZFII (yellow) and ZFIII (blue). (b) Root mean square fluctuation (RMSF) of backbone atoms (N, Cα and C) averaged over the entire course of corresponding trajectory of liganded (top panel) and unliganded (bottom panel) protein as a function of residue number. Note that the vertical boxes demarcate the boundaries of ZFI, ZFII and ZFIII within the DB domain. (c) Solvent-accessible surface area (SASA) of all atoms within each simulated structure relative to the initial modeled structure of liganded (top panel) and unliganded (bottom panel) protein as a function of simulation time.

Figure 8

Dynamic plasticity of EGR1 as probed through MD simulations conducted on the structural model of DB domain in the absence of DNA (unliganded). Shown are structural snapshots taken at 0ns, 245ns, 545ns and 801ns during the course of an MD simulation. The three tandem zinc fingers constituting the DB domain are colored green (ZFI), yellow (ZFII) and blue (ZFIII) and the coordinating zinc divalent ions have been eliminated for clarity. Note that the DNA (colored gray) bound to the 0ns snapshot is stripped away at the start of MD simulation and is only shown here for comparison.