Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis

Abstract

Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix-turn-helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel beta-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.

Figure 1

Intergenic region between cylL_L and cylR1, containing divergent overlapping promoters, P_reg and P_lyt, involved in cytolysin expression and regulation. Two palindromic sequences within the promoter region are designated IR1 and IR2. Promoter elements (−10: −10 box, −35: −35 box, RBS: ribosomal binding site, bent arrow: transcriptional start site) for P_reg and P_lyt are shown in green and red, respectively.

Figure 2

Gel shift analysis of CylR2 binding to different DNA sequences. (A) Titration of CylR2 to radiolabeled probe (1 ng probe (0.67 nM)) representing the cytolysin promoter region. (B) Effect of complementary ss IR1 oligonucleotides (p.1.s and p.1.as), alone or in combination, in order to compete for CylR2 binding to the intergenic region of the cytolysin operon (1 ng probe, 0.67 nM). (C) Competition between nested IR1 oligonucleotides shifted in the 3′ direction (2 ng probe, 1.3 nM), or (D) 5′ direction (2 ng probe, 1.3 nM) and the radiolabeled probe for CylR2 binding.

Figure 3

Stereo view of the crystal structure of CylR2. Subunits A and B are shown in red and green. Helices are labeled with α1–α5 and β-strands are labeled with β1 and β2. (A) Stereo view of the homodimer looking down the dimer two-fold axis. (B) CylR2 is rotated by 90° about the horizontal axis.

Figure 4

CylR2 in solution. (A) Correlation between experimental main-chain ¹D_NH couplings and values back-calculated from the crystal structure. (B) The ¹H-¹⁵N heteronuclear NOE plotted against the residue number. Secondary structure elements are indicated.

Figure 5

Dimer interface of CylR2. (A) Residues 1–5 and 40–65 of subunits A and B are colored red and green. Side chains of the predominantly hydrophobic residues Ile2, Pro41, Leu43, Ala46, Leu47, Lys48, Tyr51, Leu57, Phe61, Trp63 and Pro65 are shown. The orientation corresponds to Figure 3A. (B) Residues 40–60 of CylR2 (red/green) are superimposed on residues 55–74 of GerE (blue). The orientation corresponds to Figure 3B.

Figure 6

Overlay of ¹H-¹⁵N HSQC spectra of CylR2 (black) and CylR2/DNA complex (red). Black lines connect strongly shifted resonances of free and complexed CylR2. Residue Ser27 is unassigned in the complex.

Figure 7

CylR2/DNA interaction. (A) The 22-mer DNA sequence of IR1 used for NMR titration experiments. (B) Combined chemical shift changes (Δδ) for CylR2 upon addition of DNA. Chemical shift changes are shown in black for Δδ_tot, red for Δδ_CαCβ and blue for Δδ_C′. Unassigned and proline residues are labeled with an asterisk. The inset displays Δδ_tot as a solid line. Chemical shift changes for the complete amide side chains Δδ_side in magenta and for the amide C^γ or C^δ Δδ_sideC in green (see Materials and methods). Side-chain resonances not shown could not be detected. (C) Correlation between experimental main-chain ¹D_NH couplings for CylR2 in complex with DNA and values back-calculated from the crystal structure of free CylR2.

Figure 8

(A) Electrostatic surface of the CylR2 dimer with positive and negative potentials colored blue and red, respectively. (B) Chemical shift changes upon DNA binding mapped to the surface of the CylR2 dimer. Protein residues whose δ_HN and δ_N chemical shifts are affected very strongly (Y39, N40), strongly (Q29, T30, I31, Q44, L45, A46) and intermediately (Q17, S18, E19, L20) are colored orange, yellow and green, respectively. The orientation corresponds to Figure 3A.

Figure 9

LacZ activity detected from FA2-2 containing reporter constructs pLX110 (wild-type CylR2) and pLXR2N40A (Asn40 of CylR2 replaced with alanine). Data expressed in Miller units (A₄₀₅/mg protein). Cultures were either induced with FA2-2 pWH617 supernatant (containing CylL_S″) or left uninduced by incubating with FA2-2 pAT28 supernatant (vector control).

Figure 10

(A) Overall model of the CylR2/DNA complex structure. (B) Detailed stereo view indicating important protein–DNA interactions in the major groove. The DNA is shown with the sense strand in yellow and the antisense strand in cyan. Side chains of CylR2 and nucleobases interacting by van der Waals contacts and/or hydrogen bonds are indicated.