The Conformation of the N-Terminal Tails of Deinococcus grandis Dps Is Modulated by the Ionic Strength

Abstract

DNA-binding proteins from starved cells (Dps) are homododecameric nanocages, with N- and C-terminal tail extensions of variable length and amino acid composition. They accumulate iron in the form of a ferrihydrite mineral core and are capable of binding to and compacting DNA, forming low- and high-order condensates. This dual activity is designed to protect DNA from oxidative stress, resulting from Fenton chemistry or radiation exposure. In most Dps proteins, the DNA-binding properties stem from the N-terminal tail extensions. We explored the structural characteristics of a Dps from Deinococcus grandis that exhibits an atypically long N-terminal tail composed of 52 residues and probed the impact of the ionic strength on protein conformation using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism and small-angle X-ray scattering. A novel high-spin ferrous iron-binding site was identified in the N-terminal tails, using Mössbauer spectroscopy. Our data reveals that the N-terminal tails are structurally dynamic and alter between compact and extended conformations, depending on the ionic strength of the buffer. This prompts the search for other physiologically relevant modulators of tail conformation and hints that the DNA-binding properties of Dps proteins may be affected by external factors.

Keywords: DNA-binding protein from starved cells (Dps); Deinococcus grandis; Mössbauer spectroscopy; N-terminal tail extensions; biological small-angle X-ray scattering; conformational changes; mini-ferritins.

Figure 1

Biochemical characterization of purified fractions of DgrDps WT and ∆N variant. (A) SEC elution profiles of DgrDps WT (full line) and ∆N (dashed line); (B) DLS particle size distribution of DgrDps WT and ∆N variant samples; (C) progress curves of the Fe²⁺ ions oxidation reaction in the presence of O₂, for different amounts of the iron substrate (Fe²⁺/protein molar ratios of 48, 96, 192, 288 and 480). In all experiments the protein was buffered in 200 mM MOPS pH 7.0 and 200 mM NaCl.

Figure 2

Impact of the ionic strength on the structure of DgrDps WT. (A) SEC elution profiles of DgrDps WT in 50 mM MOPS pH 7.0 buffer containing varying concentrations of NaCl, between 480 mM (purple), 230 mM (blue), 150 mM (green), 80 mM (orange) and 50 mM (red), co-eluted with blue dextran for determination of the void volume. (B) Ionic strength dependence of the Stokes radius (R_S) estimated by SEC; (C) Hydrodynamic diameter (Z-average) variation with buffer salt concentration, as determined by DLS.

Figure 3

SRCD circular dichroism spectra of DgrDps WT in 10 mM MOPS pH 7.0 buffer, containing either 240 mM NaF (full circles) or 60 mM NaF (empty circles) at 25 °C. Solid line represents reconstructed spectral data from DichroWeb analysis.

Figure 4

SAXS data and ab initio modelling of DgrDps WT and ∆N variant in different ionic strength conditions. (A) Experimental scattering curves and calculated fits (solid lines) of DgrDps samples in 50 mM MOPS pH 7.0 buffer, containing varying NaCl concentrations, which are as follows: 50 mM (red), 80 mM (orange), 230 mM (blue) and 480 mM (purple) for DgrDps WT and either 50 mM (light gray) or 230 mM NaCl (dark grey) for the DgrDps ∆N variant. (B) Guinier plots and linear fits (solid lines on top of the experimental points) of the scattering profiles shown in (A); (C) pair distance distribution curves; (D) representative ab initio models of DgrDps WT (generated by GASBOR) and ∆N (generated by DAMMIN) in the different sample conditions tested, superimposed with the dodecamer model for each protein (ribbons).

Figure 5

Mössbauer spectra of ferrous iron loading experiments for DgrDps WT and ∆N. Samples of proteins apo-form were incubated with different molar ratios of ferrous ⁵⁷Fe (from 6 to 48 Fe per protein, WT in spectra (A–D) and ∆N in spectra (E–H) in anaerobic conditions for 20 min. Samples were prepared in buffers containing 200 mM MOPS pH 7.0 and 200 mM NaCl. The spectra were recorded at 80 K, with no external magnetic field applied. The solid lines are theoretical simulations using the parameters listed in Table 3. Site I, II, and additional ferrous species are presented in blue, red, and green, respectively.

Figure 6

Occupancy of each type of ferrous iron in the iron-loaded Mössbauer samples of DgrDps WT (A) and ∆N variant (B). The deconvolution of each spectrum into three different ferrous species allows the determination of the area percentage of each signal, which in turn can be used to calculate the stoichiometry of each type of ferrous signal per protein, considering the total amount of Fe per protein added. Site I, II, and additional ferrous species are in blue, red, and green, respectively.