Structural insights into DNA binding domain of vancomycin-resistance-associated response regulator in complex with its promoter DNA from Staphylococcus aureus

Abstract

In Staphylococcus aureus, vancomycin-resistance-associated response regulator (VraR) is a part of the VraSR two-component system, which is responsible for activating a cell wall-stress stimulon in response to an antibiotic that inhibits cell wall formation. Two VraR-binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N-terminal receiver domain (VraR_N ) connected to a C-terminal DNA binding domain (VraR_C ) with a flexible linker. Here, we present the crystal structure of VraR_C alone and in complex with R1-DNA in 1.87- and 2.0-Å resolution, respectively. VraR_C consisting of four α-helices forms a dimer when interacting with R1-DNA. In the VraR_C -DNA complex structure, Mg²⁺ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg²⁺ to VraR_C enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraR_C with R1- and R2-DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic-resistant S. aureus via targeting VraR.

Keywords: DNA binding domain; NMR; Staphylococcus aureus; response regulator; two-component system; vancomycin resistance; x-ray crystal structure.

FIGURE 1

AUC‐SV profiles showing sedimentation coefficient distribution. All the data were analyzed by using Sedfit for assessing molecular weights. (a) The c(S) distribution of unbound R1‐DNA is at 2.69 S and active VraR in complex with R1‐DNA had a peak shift at 5.07 S. (b) The c(S) distribution of inactive VraR and unbound R1‐DNA are at 2.7 S and a minor peak at 5.2 S for inactive VraR–DNA complex

FIGURE 2

Structural overview of VraR_C in complex with promoter DNA. (a) Upstream and downstream binding of two VraR_C subunits binding to R1‐DNA are shown in blue and orange, respectively. Secondary structural elements on both the subunits are labeled and six Mg²⁺ ions are in green spheres. (b) Schematic diagram showing detailed interactions between two VraR_C molecules and DNA. Hydrogen bonds, electrostatic interactions are shown in green and brown dotted lines

FIGURE 3

Superimposition of VraR_C from the R1‐DNA complex in yellow and activated VraR DBD (PDB ID: 4IF4) in light‐cyan. The Subunit‐B has slight rotation upon binding to DNA. Lys177 and Lys180 in the VraR_C dimer from α9 helices are involved in binding to DNA are shown as sticks. Distances between two Lys177 and two Lys180 residues within DNA‐bound VraR_C dimers were 2.2 and 1.0 Å closer than active VraR

FIGURE 4

(a) The overlaid 2D ¹H, ¹⁵N TROSY‐HSQC spectra were acquired from inactive VraR (blue) and VraR_C (red). Residues Gln204 and Glu191 of α10 helix that interact with the N‐terminal domain of inactive VraR are labeled on VraR_C. (b) The overlaid 2D ¹H, ¹⁵N TROSY‐HSQC spectra were acquired from active VraR (blue) and VraR_C (red). Residues Gly162, Thr196 and Gln197 involved in the DBD dimer of active VraR are labeled on VraR_C. All samples were prepared at pH 6.0 and acquired spectra at 298 K

FIGURE 5

Interaction of VraR_C with both R1‐ and R2‐DNAs inspected by NMR titrations. Plots of the normalized chemical shift perturbations of VraR_C after binding to R1‐DNA (a) and R2‐DNA (b). Gaps indicate the missing peaks from DNA titration. Perturbed residues with values >0.06 (dotted black line) mapped on the structure of VraR_C for both (c) R1‐DNA and (d) R2‐DNA titrations

FIGURE 6

Mg²⁺ ions shown as green spheres binding to Asp194 of two subunits in the VraR_C–DNA complex. Subunits are shown in orange and blue. Each Mg²⁺ ion has a hydrogen bond interaction to the OD2 atom of Asp194 with a distance of 2 Å and 2.1 Å. Five water molecules coordinating the Mg²⁺ ions are in red spheres. The 2Fo‐Fc electron density map of Asp194, water molecules and Mg²⁺ ions was contoured at 1σ and shown as a blue mesh

FIGURE 7

R1‐DNA binding affinity of VraR_C at different concentrations measured with BLI technology both in the absence (a) and presence (b) of 10 mM MgCl₂. All data were processed and analyzed by a double reference subtraction method by using Octet Data Analysis Software. The 2:1 binding model of association and dissociation function was used to determine K _d in the global fitting. (c) The electrostatic potential surface of VraR_C was calculated with the Pymol plugin APBS, showing red for the negative surface and blue for the positive charge surface. A slight negative charge appearing near the side chain of Asp194 was labeled. (d) Thermal denaturation curves of VraR_C–DNA complex in the presence (blue) and absence (red) of 10 mM MgCl₂ were monitored at 222 nm, revealing melting temperatures of 61.0 and 56.3°C, respectively

FIGURE 8

Packing within the crystal of VraR_C–DNA complex is shown among three asymmetric units in pink, blue and yellow. Crystal contacts of DNAs were observed between two C₄–C₄ and C_11′–C_11′ nucleotides. The detailed Mg²⁺ ions interaction within two asymmetric units between DNAs are in boxes. Each Mg²⁺ ion (green spheres) binds with OP1 (asymmetric unit1) and OP2 (asymmetric unit2) atoms of C₄ nucleotides with 2.0 and 2.1 Å and are surrounded by water molecules (red spheres)