Structural insights into dynamics of RecU-HJ complex formation elucidates key role of NTR and stalk region toward formation of reactive state

Abstract

Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state.

Figure 1.

Crystal structure of inactive mutant of RecU (RecU_D88N)–DNA complex. (A) Crystallographic asymmetric unit (ASU) in crystal structure of RecU–DNA complex consists of four RecU monomers forming two dimers bound to two DNA duplexes. Cartoon representation of protein Chains A, B, C and D are shown in green, cyan, pink and yellow respectively. Chain E, F, G and H of DNA are shown in orange, limon, blue and red respectively. (B) Interactions of RecU with DNA duplexes: Cap and stalk regions are shown in white and black, respectively. Hydrogen bonding and active site residues are shown as van der walls spheres, while the DNA is shown as stick. Residues which form hydrogen bonds in all four monomers of RecU in ASU are shown in blue. Residues that form hydrogen bonds in only some of monomers are shown in yellow. Active site residues are shown in Red. (C) Distance between Cα atoms of residues R71 of one monomer and D88 of its dimerising partner (binding pocket) for both AC and BD dimer are shown for the structure presented here and for the structure of B. stearothermophilus. (D) Electron density (2F_o – F_c map) is shown at sigma level of 0.7 for the 33 residue long NTR region of chain D. the sphere representation is shown for the NTR coloured red. The ASU is coloured green while symmetry mates forming the crystal contacts are coloured white.

Figure 2.

SAXS analysis of DNA free RecU (D88N). (A) Fit of Ensemble Optimized Modeling (shown in red) of SAXS data to the experimental SAXS data (shown in blue). The χ²-value of the fit is 1.89. (B) Ensemble of conformations of NTR generated from EOM analysis are shown in different colors. The core structure of the RecU enzyme is shown in magenta. Completely extended conformation of NTR are shown in yellow and red, partially collapsed conformation in green and cyan, and completely collapsed conformation of NTR is shown in white.

Figure 3.

Thermofluor studies. (A) Thermofluor experiments were carried out to study the induced stabilization of RecU on its binding to different lengths of HJ and DNA duplex. Holliday junctions formed by the oligomers of lengths 9 bp, 10 bp, 11 bp, and 12 bp were used. DNA duplex used is same as that of the crystal structure presented here. RecU in the absence of any DNA was used as control. Thermofluor experiments for each of these systems were carried out in quadruplets. T_m was calculated from first derivatives of fluorescence intensities with respect to temperature. The scatter plot gives a clear picture of stabilization of RecU on HJ binding. (B) Unfolded fractions as a function of temperature for RecU_D88N and its complex with HJ of an arm length of 12 bp. (C) Conformational entropy calculated from the profiles in (B).

Figure 4.

Principal Component analysis. (A) Projection of wild type RecU (RecU_WT: shown in black), ΔNTR mutant RecU (RecU_ΔNTR: shown in red) and their complexes with HJ i.e. (RecU_HJ: shown in green), (RecU_ΔNTR-HJ: shown in blue) along the first two eigenvectors. Sampling along both these eigenvectors is restricted in the presence of HJ. (B) Motion along eigenvector 1 showing rotation of mushroom cap with respect to the the stalk region. Two extreme structures along the direction of the eigenvector are shown in red and blue respectively in a surface representation of a backbone trace. (C) Motion along eigenvector 2 showing rocking of mushroom cap with respect to the stalk region.

Figure 5.

Mechanism of cleavage of phosphodiester bond at the RecU active site. (A) Plausible mechanism of DNA cleavage by RecU based on the mechanism reported earlier (64). Mg⁺² will cause polarization of water molecules around it. Carboxylic oxygen of E101 (one that is not coordinated with Mg⁺²) can deprotonate a water coordinating with Mg⁺². The deprotonated water will then attack the phosphate giving rise to penta-coordinated phosphate intermediate that is stabilized by Lewis acid K103. Another Mg⁺² coordinated water can then act as general acid by protonating the leaving anion. (B) Percentage occurrence of step 1 of the above mechanism observed in the simulation of RecU in complex with holliday junction (HJ) (RecU_HJ) and ΔNTR mutant in complex with HJ (RecU_ΔNTR-HJ). (C) Atomic model of step 1 of proposed mechanism as observed in simulation (one such snapshot). DNA and active site residues are shown in stick representation. Water and Mg⁺² are shown as spheres and are coloured in red and green respectively. (D) Projection of simulation-2 of RecU_HJ (for which the occurrence of step 1 of the proposed mechanism is highest) along eigenvector 1 and 2. RecU_WT: shown in black; RecU_ΔNTR: shown in red; RecU_HJ: shown in green; RecU_ΔNTR-HJ: shown in blue; simulation 2 of RecU_HJ: shown in orange; projection of X-ray structure is shown as a triangle.

Figure 6.

Plausible mechanism of binding and insertion of RecU in HJ. (A) RecU and a Holliday junction (HJ). (B) RecU in the unbound form exhibits rocking motion as described by the Ev2. With this domain motion RecU inserts itself in the HJ. (C) RecU-HJ complex is formed. (D) RecU then searches for the cleavage site by the rotation of the cap domain (motion observed along eigenvector Ev1). (E) Reactive state in the active site is then formed due to the breathing motion of mushroom cap. This breathing motion was observed in the functional mode analysis on the occurrence of the reactive state. Once reactive state is formed, phosphodiester bond cleavage occurs. (F) Cleaved HJ then diffuses from the RecU.