Conformational dynamism for DNA interaction in the Salmonella RcsB response regulator

Abstract

The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a β5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic.

Figure 1.

Dimerization of full-length RcsB bound to BeF₃⁻. (A) Cartoon representation of the dimerization surface of RcsB bound to the phosphomimetic BeF₃⁻ (labeled as BeF) through the REC domain (blue and cyan) and DBD domains (yellow shades). Surface of the REC domains is shown. The REC–DBD connector is colored in salmon. (B) A framed detail of the dimerization surface and catalytic center in the dimer showing relevant residues, BeF₃⁻, Mg⁺² ion (Mg). (C) Detail of the active site in one of the subunits showing interactions by relevant residues, BeF₃⁻ (BeF) and Mg⁺² (Mg) ion and water molecules (W) as dashed lines. (D) Autophosphorylation of RcsB with 50 mM AcP is visualized using different techniques (Phostag-acrylamide, autoradiography and native-PAGE). (E) RcsB dimer formation upon phosphorylation is visualized by gel filtration with 200 μg of purified protein using a Superdex200 increased 10/300GL (GE, Healthcare)

Figure 2.

Model of the interaction of RcsB with DNA. (A) Superposition of the DBD domains in RcsB_BeF structure (in yellow) with the DBD NarL structure bound to DNA (PDB ID: 1ZG1) (in blue). (B) Side chain for residues Lys154, Lys180, Gln185 and Lys187 are shown as sphere in magenta. A framed detailed view of same residues in RcsB interacting with DNA is shown (C) Sequence alignment of RcsB and NarL comprising REC–DBD connector (in salmon) and DBD (yellow). Mutated residues at the DBD in RcsB involved in interactions are highlighted in magenta. (D) EMSA assays with RcsB WT and mutants in the absence and presence of phosphorylation (with 50 mM AcP) and P1_flhDC site (−228 to −203) in the flhDC operon.

Figure 3.

Structure of RcsB in the absence of BeF₃⁻. (A) RcsB hexamer structure, in the absence of BeF₃⁻, composed by three dimers related by a 3-fold axis resembling a cylinder. The bases of the cylinder are generated by the REC domains and DBDs are at the central part of the cylinder. (B) Dimer crossed conformation of RcsB (RcsB_crossed; in dark pink and purple) with a sulfate ion (SO₄²⁻ labeled as SO₄) bound at the active site (on the left) shows dimerization mainly through REC and DBD from different subunits. Dimer structures of the S207C-RcsB mutant in the crossed conformation at the middle (S207C–RcsB_crossed; in pink and blue) and in an alternative asymmetric crossed dimer conformation on the right (S207C–RcsB_AC; in violet and orange) (C) Formation of RcsB_crossed dimers with mutant S207C in the absence and presence of 0.1 mM of CuPh is observed in SDS–PAGE (left panel). EMSA experiments with WT and mutant S207C in absence and presence of 50 mM AcP and 0.1 mM CuPh (right panel) (D) In this conformation, RcsB can bind two separate dsDNA fragments (in gray) (E) Model of the RcsB hexamer wrapped by two separate dsDNA fragments.

Figure 4.

Active site and functional studies on catalytic residues in RcsB. (A) Detail of the active site for RcsB_crossed (in pink) superposed with RcsB_BeF (in cyan). Catalytic residues as well as the sulfate ion (SO₄²⁻ labeled as SO₄) and BeF₃⁻(labeled as BeF) are shown as sticks. (B) Another view of the active site in RcsB_crossed (pink for one subunit and purple for the other). Residue E170 (in purple) from the DBD of the other subunit contributes to the active site. Catalytic and relevant residues are shown as sticks together with the sulfate ion (SO₄) and a water molecule (W). (C) Phosphorylation assays of WT and mutants of RcsB with AcP³². Phosphorylation was followed at 5, 10, 20, 40 and 60 min and was evaluated with the MultiGauge software (Fuji). (D) EMSAs of RcsB WT and mutant forms with P1_flhDC were performed in the absence and presence of 50 mM of AcP.

Figure 5.

Effect of RcsB mutations in capsule formation and motility. Overexpression of RcsB mutants in the Salmonella Typhimurium strain MD4821 (igaA1 rcsB) to monitor effect on colanic capsule production and motility in vivo. (A) Expression of the WT, unphosphorylatable mutant D56A and empty vector (as control). (B) Expression of mutants at residues present in the DBD domain that either interact with DNA according to the model RcsB_BeF–DNA or contribute to DBD–DBD dimerization (L202). (C) Expression of mutants at residues that contribute to specific interactions between REC and DBD at each subunit. (D) Expression of mutants at relevant residues H12 and M88 that contribute to RcsB dimerization and catalysis. (E) Expression of mutants at residue S207C that traps DBD–DBD in the crossed conformation due to disulfide bond formation. A double mutant (S207C/M88A) containing an additional mutation M88A is also shown.

Figure 6.

Switch mechanism (β5-T coupling) of RcsB between the inactive and active conformation. (A) Comparison between the unphosphorylated (PDB ID: 5I4C in green) and phosphorylated conformation (RcsB_BeF in cyan) reveals movements, induced by the presence of β6, in the switch residues T87 and L108 while I106 shows the similar inward conformation. (A and B) These movements produce conformational changes, mainly in Lβ4α4 and Lα5β5, that allow α1 and Lβ1α1 from the other subunit (in magenta) to insert H12 for RcsB dimerization. (B) The movements of Lβ4α4 and Lβ5α5 are necessary to avoid clashes between H12 (in magenta), Q110 at Lβ5α5 and M88 at Lβ4α4. Additionally, movement of Lβ4α4 avoids clashes between N90 and α6 at the REC–DBD connector.

Figure 7.

Model for RcsB DNA-binding regulation mediated by phosphorylation. The initial activation of the Rcs phosphorelay triggers RcsB phosphorylation, which dimerizes through the α1–α5 surface. This asymmetric dimer is able to bind, with high-affinity, DNA sequences with two half-sites, mainly with tail-to-tail arrangement, producing a fast short-term response. If signal persists, an excess of highly dynamic phosphorylated RcsB molecules could form crossed dimers able to bind a second pool of DNA sequence with different features and acquire the hexameric architecture inducing DNA supercoiling, producing a long-term response. We suggest that the asymmetric dimer could show a slow dephosphorylation as dimerization locks the active site hindering the release of the phosphoryl group while the crossed dimer could dephosphorylate faster, as the active site is more solvent exposed. We cannot exclude that RcsB could fluctuate between the asymmetric and crossed conformations (represented by dashed gray lines), during its regulation, reflecting its conformational dynamism.