Plasticity and Co-Factor-Dependent Structural Changes in the RecA Nucleoprotein Filament Studied by Small-Angle X-Ray Scattering (SAXS) Measurements and Molecular Modeling

Abstract

Structural analyses of protein filaments formed by self-assembly, such as actin, tubulin, or recombinase filaments, have suffered for decades from technical issues due to difficulties in crystallization, their large size, or the dynamic behavior inherent to their cellular function. The advent of cryo-electron microscopy has finally enabled us to obtain structures at different stages of the existence of these filaments. However, these structures correspond to frozen states, and the possibility of observations in solution is still lacking, especially for filaments characterized by a high plasticity, such as the RecA protein for homologous recombination. Here, we use a combination of SAXS measurements and integrative modeling to generate the solution structure of two known forms of the RecA nucleoprotein filament, previously characterized by electron microscopy and resolved by X-ray crystallography. The two forms differ in the cofactor bound to RecA-RecA interfaces, either ATP or ADP. Cooperative transition from one form to the other has been observed during single-molecule experiments by pulling on the filament but also in solution by modifying solvent conditions. We first compare the SAXS data against known structural information. While the crystal structure of the ATP form matches well with the SAXS data, we deduce from the SAXS profiles of the ADP-form values of the pitch (72.0 Å) and the number of monomers per turn (6.4) that differ with respect to the crystal structure (respectively, 82.7 Å and 6.0). We then monitor the transition between the two states driven by the addition of magnesium, and we show this transition occurs with 0.3 mM Mg ²⁺ ions with a high cooperativity.

Keywords: RecA nucleofilament; SAXS; helical protein assembly; integrative modeling; protein filament plasticity.

Figure 1

Estimating the filament length for the extended (A,B) and the compressed (C,D) forms of the RecA nucleoprotein filament. Panels (B,D) show a close-up of the curves in (A,C), restricted to q values greater than $0.05 {\AA }^{-1}$. The experimental SAXS signal (open black circles) is shown together with theoretical scattering curves obtained using FoxS from filament models of different lengths constructed from the crystal structures with PDB code 3CMW (A,B) and 2REB (C,D). The inserts in panels (A,C) provide the color codes associated with the different filament lengths. The insert in panel (A) applies to panels (A,B); the insert in (C) applies to panels (C,D). Intensities I(q) are in ${\mathrm{cm}}^{-1}$.

Figure 2

Scattering curves for (A,B) extended and (C–F) compressed forms of the RecA nucleoprotein filament. The experimental curve is represented with black circles, while curves reconstructed from models using SAXS are shown with colored lines. Color codes associated with (P, N) values are provided as inserts of panels A (A,B), C (C,D), and E (E,F). In panels (B,D,F), only the region of the curve with angle values ranging from 0.05 to $0.15 {\AA }^{-1}$ is shown, emphasizing the variability in the position of the first minimum ${\mathrm{q}}_{min}$ and the first maximum ${\mathrm{q}}_{max}$. Intensities I(q) are in ${\mathrm{cm}}^{-1}$.

Figure 3

Correlations between (A) the number of monomers per turn and (B) and the pitch values and the position of ${\mathrm{q}}_{min}$ (blue squares) and ${\mathrm{q}}_{max}$ (red circles). The best-fitted thin lines are colored with the same color codes. Horizontal broken straight lines, blue for ${\mathrm{q}}_{min}$ in the (A) panel and red for ${\mathrm{q}}_{max}$ in the (B) panel indicate the experimental ${\mathrm{q}}_{min}$ and ${\mathrm{q}}_{max}$ values.

Figure 4

Structure and electrostatic properties of five helical turns of extended (left) and compressed (middle,right) forms of the RecA filament that correspond to the SAXS data. Electrostatic isosurfaces close to the protein surface, with values of −1 and 1, are represented in red and blue, respectively. Electropositive potential accumulates in the wide filament groove of the extended form, forming continuous tracks. Conversely, the compressed form presents a straighter groove, with discontinuous patches of electropositive and electronegative potential. The electrostatic potential was calculated in 0.15 mM NaCl using the APBS and PDB2PQR softwares (respectively version 3.4.1 and 3.6.1) via the webserver https://server.poissonboltzmann.org/, accessed on 14 April 2025, [44], and visualized using the Visual Molecular Dynamics (VMD) software [45], version 1.9.4. The right panel displays a cartoon structure of the compressed filament with bound DNA and ATP in the absence of magnesium.

Figure 5

${\mathrm{Mg}}^{2+}$ titration of a RecA–ATP$\gamma$S–DNA nucleofilament. (A) Experimental scattering curves obtained at increasing ${\mathrm{Mg}}^{2+}$ concentration; the color code is indicated as an insert; (B) details of the curves for q values between 0.05 and $0.12 {\AA }^{-1}$; (C) evolution of the ln(I(q)) values taken at q = $0.07 {\AA }^{-1}$ as a function of the magnesium concentration. Intensities I(q) are in ${\mathrm{cm}}^{-1}$.