Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA

Abstract

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-A crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix-turn-helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122 degrees rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.

Figure 1.

Key structural features of YvoA from Bacillus subtilis. (A) Crystal structure of the YvoA homodimer (orange/cyan) at 2.4-Å resolution with bound sulfate ions. Residues/positions susceptible to proteolytic cleavage are marked in red and purple. (B) Topology plot of the YvoA monomer. Circle and asterisk mark the sulfate-binding sites 1 and 2, respectively. (C) Ribbon representation of the effector-binding domain highlighting the internal 2-fold rotational symmetry of the fold. The 2-fold axis passes through the sulfate ion that is hold in place by helices α5 and α9. (D) Stereo view of the superimposition of the effector-binding domain of YvoA (orange) and chorismate lyase from E. coli (in green, PDB code: 1G1B) (14).

Figure 2.

GlcNAc-6-P effector binding to YvoA. (A–C) Binding isotherms of YvoA and YvoA mutants (0.9 mg ml⁻¹) titrated with 10 mM GlcNAc-6-P. (A) Wild-type YvoA, (B) YvoA-I209E and (C) YvoA-A224R. (D) Stereo view showing the proposed effector-binding site of YvoA. GlcNAc-6-P in β-configuration can be docked into weak non-protein electron density present in the initial MAD-phased experimental electron density map near monomer B (in blue). (E) Schematic representations of the interactions between GlcNAc-6-P and side-chains from the effector-binding site of YvoA (chain B). GlcNAc-6-P is not part of the deposited refined crystal structure and is therefore colored transparently. The position of the sulfate coincides with that of the phosphate group of GlcNAc-6-P.

Figure 3.

DNA-binding properties of YvoA. (A) Gel filtration of YvoA (solid line), upon addition of equimolar dsDNA (dashed line), and with YvoA in excess over dsDNA (dotted line) demonstrating clear shifts of the elution peaks as a function of the protein:DNA ratio. (B) Ribbon representation of a DNA-bound YvoA model. The composite model was generated by modeling YvoA according to the conformation observed in YydK (PDB code: 3BWG) and superimposed on the DNA-binding heads of the DNA-bound wHTH domains of FadR (PDB code: 1HW2) (35). (C) Sequence alignments of the DNA-binding domains of YvoA from B. subtilis, FadR from E. coli, and YydK from B. subtilis. The secondary structure elements, indicated by helices and arrows, refer to YvoA. Residues conserved between YvoA and FadR (and partially YydK) are highlighted by blue shaded boxes. Residues conserved only between YvoA and YydK are indicated by cyan shaded boxes. Residues implicated in DNA binding in FadR (35) are highlighted by red boxes.

Figure 4.

Models of disulfide-bridged YvoA mutants and their DNA-binding properties. (A) Model of the homodimeric cystein mutant YvoA-E61C-L242C viewed from two different angles. This model is supposed to represent the DNA-bound conformation leading to repression of YvoA-controlled genes. The inset shows the region around the intermolecular disulfide bridge between C61 and C242′. (B) Model of the homodimeric cystein mutant YvoA-K24C-G97C resembling the induced conformation. The inset shows the region around the intramolecular disulfide bridge between C24 and C97. (C) Left: gel filtration of free wild-type YvoA (black), YvoA-E61C-L242C (blue), and YvoA-K24C-G97C (red) reveals only small differences in the elution profile. Right: gel filtration of YvoA variants in the presence of dsDNA (18mer) shows major peak shifts. The black line represents wild-type YvoA in the presence of equimolar dsDNA. The blue line shows YvoA-E61C-L242C in excess over dsDNA. The red line represents YvoA-K24C-G97C in the presence of equimolar dsDNA.

Figure 5.

Limited proteolysis assay of YvoA. YvoA (0.35 mg ml⁻¹, i.e. 6-µM dimer) was incubated for 180 min with 0.014 U mg⁻¹ subtilisin in the presence or absence of dsDNA and/or GlcNAc-6-P. ‘Double dagger’, ‘dagger’, ‘asterisk’ and ‘section’ mark cleavage products starting with the N-terminal residues K5, R96, L86 and R70, respectively. Molecular mass estimates suggest that the fragments R96, L86 and R70 result from a single cleavage event. This is not the case for the K5 fragment, which is further truncated toward the C-terminus. For complete time courses see Supplementary Figure S6.

Figure 6.

Induction mechanism of YvoA. (A) Stereo representation of the allosteric rearrangement in YvoA upon effector binding. The model in the DNA-bound conformation is shown in grey, the crystal structure of induced YvoA in orange. The interdomain loop in the DNA-bound model is highlighted in blue. Upon effector binding, this loop switches conformations and folds into helices α4 and α5 (colored in red). As a consequence, the DNA-binding domain rotates as a rigid body by 122° around the indicated axis. (B) Magnified stereo view of helices α4 and α5 in induced YvoA. The σ_A-weighted 2F_o–F_c electron density map shows well-defined, continuous density for the interdomain linker in YvoA. (C) Allosteric mechanism of YvoA induction. In the DNA-binding conformation (in gray), homodimeric YvoA binds DNA with its DNA recognition helices paired together at the dimer interface and binding into successive major grooves. In doing so, YvoA is able to block transcription of any downstream genes. Upon effector (GlcNAc-6-P) binding, the interdomain linker region undergoes a loop-to-helix transition forcing the DNA-binding domains apart in a ‘jumping jack’-like motion (orange). YvoA is then not able any more to bind to the same dsDNA duplex with both DNA-binding domains simultaneously, causing de-repression of YvoA-controlled genes.