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. 2015 Mar 18;10(3):e0116414.
doi: 10.1371/journal.pone.0116414. eCollection 2015.

An Integrative Approach to the Study of Filamentous Oligomeric Assemblies, With Application to RecA

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Free PMC article

An Integrative Approach to the Study of Filamentous Oligomeric Assemblies, With Application to RecA

Benjamin Boyer et al. PLoS One. .
Free PMC article

Abstract

Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called "extended" and "compressed" forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Overview.
Binding geometries generated by a PTools/ATTRACT coarse-grained docking simulation are analyzed with Heligeom in terms of the helical parameters of regular assemblies that they define. The results are filtered based on relative energies and geometry considerations (see S1 Fig, supporting information). Binding geometries with near-cyclic organizations but suffering from steric clashes are submitted to an optimization process in which they are adjusted towards the two closest cyclic geometries (S1 Protocol, supporting information). Binding geometries leading to steric clashes that are also not in the near-cyclic category are currently not analyzed further but may be adjusted to helical organization in future developments. Heligeom can be applied to any of the final structures to construct filamentous or cyclic assemblies of arbitrary size for further analysis.
Fig 2
Fig 2. RecA-RecA docking.
(A) Superposition of monomers taken from the 2REB (blue) and 3CMW (red) crystal structures, in cartoon representation; the rigid core is the region where the two structures are almost superimposable. (B-D) Binding regions on the surface of one RecA monomer, restricted to its rigid core. The left side shows the top of the monomer, the right side the bottom. (Note: orientations are chosen to best show the interacting surface, and are not exactly 180 apart). (B-C) Interface patterns characterizing the X (B) and the X* (C) binding modes. In both panels B and C, the union of the two interfaces is shown in pale yellow, while the specific X or X* interface is overlaid in blue (B) or orange (C). (D) Each residue is colored according to the best interaction energy of the interfaces to which it belongs, normalized by the best interaction value found in the simulation. The color ranges from blue to red to indicate 0–100% of the maximum interaction value. The interaction energies result from merged docking simulations carried out using the 2REBcore or the 3CMWcore monomers (see Methods). The rigid core represented as a 3D support in all views is 2REBcore.
Fig 3
Fig 3. RecA self-assembly.
Modes of regular association of RecA monomers resulting from docking simulations with ATTRACT. Structures labeled A to E represent cyclic assemblies, structure F is nearly straight, structure G is a left-handed helix and structures H, I are right-handed helices corresponding closely to the 2REB [21] and 3CMW [23] crystal structure forms (binding modes X and X*), respectively. Complete Heligeom characterizations of these structures are provided in Table 2.
Fig 4
Fig 4. Exploration of the RecA-ADP (X) and RecA-ATP (X*) structural families.
Helical characteristics of samples obtained via bound-bound docking simulations targeted to particular binding modes X (left) and the X* (right) followed by Monte Carlo exploration (see Methods). Sampled geometries are represented by the number of monomers per turn (N) versus pitch (in Å). The results are colored according to the interaction energy E in RT units, with E ≤ −42 (orange); −42 < E ≤ −40 (red); −40 < E ≤ −38 (green); −38 < E ≤ −36 (blue); E > −36 (cyan). MC sampling runs starting from the exact X or X* binding modes as extracted from the corresponding crystal structures are indicated by arrows. Inserts show representative sampled structures for each binding mode. As an indication of the degree of correspondence between the sampled geometry and the targeted binding mode, angular deviations (calculated using Heligeom) from the targeted binding geometry X or X* for the representative samples labeled 1, 2, 3 are 14.2, 5.2 and 23.3 (from X) and 5.1, 11.0 and 9.3 (from X*), respectively. As angular deviations are measured with respect to a local screw axis in each case, only their absolute values are globally meaningful.
Fig 5
Fig 5. View of one turn of a RecA negatively supercoiled filament at atomic resolution.
The structure has been constructed following the geometric characteristics described by Shi and collaborators [18], with a pitch P = 160 nm for the the supercoil (see text and S1 Protocol, supporting information). The axis of the two strands (in cyan and green, respectively) are separated by 110 Å. The DNA incorporated in each of the two RecA filaments is in red.
Fig 6
Fig 6. Models of RecA filaments with alternating binding modes.
Consecutive monomers are represented in surface representation with different colors. (A) The model features a junction between X*-form regions (monomers 1–18) and X-form regions (18–36). (B) The filament, principally in the X* form, presents X-type interfaces periodically distributed every 6 monomers. (C) 24-mer filament with alternating X* and X interfaces. The monomers with an X-type upper interface are represented in green, those with an X*-type upper interface in cyan. Pitch values characterizing the helices in A and C and the superhelix in B are indicated.

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Grant support

The authors acknowledge support from Université Paris 6 (to B.B.), the French CNRS (Centre National de la Recherche Scientifique, to J.E.), IFCPAR (Indo-French Center for the Promotion of Advanced Research, to C.H.R.), the German DFG (Deutsche Forschungsgemeinschaft, within SFB749, project C5, to M.Z.), and the “Initiative d’Excellence” program from the French State (Grant “DYNAMO”, ANR-11-LABX-0011-01, to C.P. and C.H.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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