Structural insights into RapZ-mediated regulation of bacterial amino-sugar metabolism

Abstract

In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.

Figure 1.

The role of GlmY/GlmZ sRNAs in regulating glucosamine-6-phosphate (GlcN6P) levels in Escherichia coli. (A) Predicted secondary structures of the sRNAs GlmY and GlmZ (8). The sites that are processed to generate the mature forms of these sRNAs are indicated by orange arrows. GlmZ nucleotides involved in base-pairing with the glmS transcript are labelled in red. (B) Model for the control of the GlmY/GlmZ cascade by RapZ. When GlcN6P concentrations are high in the cell, GlmY is present in low amounts. Under these conditions, RapZ is free to bind GlmZ and facilitate processing by RNase E, thereby inactivating GlmZ and blocking GlmS synthesis. When GlcN6P levels decrease, processed GlmY accumulates and binds and sequesters RapZ. As a result, GlmZ is free to base-pair with glmS in an Hfq-dependent manner and activate synthesis of GlmS, which catalyses the generation of GlcN6P. Figure adapted from Göpel et al. (8).

Figure 2.

A protease-resistant RapZ C-terminal region forms a structurally autonomous domain that homodimerises. (A) Limited proteolysis reveals a truncated species resistant to digestion. Full-length RapZ (1 mg/ml) was digested by trypsin (0.02 mg/ml) over the course of 75 min at 37°C, revealing a truncated species resistant to digestion spanning residues 154–284, as confirmed by mass spectrometry (data not shown). This residue range corresponds to the C-terminal RNA-binding domain of RapZ. The red asterisk denotes a band corresponding to trypsin used for digestion. (B) SEC-MALS analysis (left panel) reveals that RapZ-CTD elutes as a single peak on the chromatogram. Analysis of the peak fractions is summarized in the right panel. (C) AUC analysis in the sedimentation velocity mode of RapZ-CTD. The left and right panels show distributions for sedimentation coefficient (c(S)) and molecular mass (c(M)), respectively.

Figure 3.

The X-ray crystal structure of the C-terminal domain of RapZ. (A) The X-ray crystal structure of RapZ-CTD in two orientations shown as cartoon representation. The two RapZ-CTD protomers are coloured blue and red. (B) The RapZ-CTD dimer shown as an electrostatic surface representation. Two views as in (A), with electropositive regions coloured in blue and electronegative regions coloured in red. Electropositive residues previously predicted to be involved in RNA binding (8) are labelled on one protomer of the RapZ-CTD dimer. (C) The malonate binding pocket of the RapZ-CTD. Top: a protomer of RapZ-CTD shown in the same orientation as 3A, as a red cartoon with residues contacting the bound malonate shown as sticks. Bottom: a close up view of a putative ligand-binding pocket that is occupied by malonate. Malonate is shown as blue sticks, with a F_o-F_c omit map for the malonate shown as grey mesh contoured at 3 σ. Residues coordinating the malonate are shown as red sticks, and water molecules are shown as red spheres. Hydrogen bonds are represented by grey dashed lines.

Figure 4.

The X-ray crystal structure of RapZ. (A) The X-ray crystal structure of RapZ in three orientations in cartoon representation with semi-transparent surface. The RapZ protomers are coloured red, blue, orange and green. (B) Electrostatic surface representation of the RapZ tetramer in three orientations as in A. Electropositive regions are coloured blue and electronegative regions are coloured red. (C) Diagram representation of the global domain architecture of RapZ. The protomers of RapZ are coloured red, blue, orange and green as in A. The protein assumes a tetrameric assembly in the crystal structure wherein some NTD-CTD contacts are satisfied whereas others are not. It is formally possible that in solution the tetramer is reorganized as a self-closing dimer of dimers that satisfies all the NTD-CTD contacts. (D) SEC-MALS analysis (top) reveals that RapZ elutes as a single peak in the chromatogram. Analysis of the peak fractions is summarized in the bottom panel. (E) A close up view of the putative Walker A-box in a single protomer of RapZ. Bound sulfate is shown as yellow and red sticks, with a F_o-F_c omit map for the ligand shown as grey mesh contoured at 3 σ. Residues forming the Walker-A box are shown. Hydrogen bonds are represented by grey dashed lines. (F) Sulfate modelled bound at the ‘malonate’ binding site in the RapZ-CTD (In the same orientation as Figure 3C). Bound sulfate is shown as yellow and red sticks, with a F_o-F_c omit map for the ligand shown as grey mesh contoured at 3 σ. Residues coordinating the ligand are shown as red sticks. Hydrogen bonds are represented by grey dashed lines.

Figure 5.

Self-interaction of RapZ domains in vivo. (A) RapZ NTD and CTD in isolation can form homo-oligomers and they also cross-interact in BACTH assays. Strains carrying plasmids encoding for the T18- and T25- fusion proteins as indicated in the left panel were grown to stationary phase and the β-galactosidase activities were measured. Plasmids encoding T25 or T18 domains alone served as a negative control. The right hand panel shows a schematic representation of the RapZ tetramer as seen in the crystal structure, and the individual domain interactions probed in this assay. (B) The β-strand comprising residues 179–182 is essential for self-interaction of the RapZ-CTD as revealed by BACTH. β-Galactosidase activities of strains producing variants of the RapZ-CTD fused to both, the T25- and T18-fragments of CyaA are shown in the left panel. The right panel shows the RapZ tetramer structure, with the CTD:CTD interaction probed in this assay highlighted in the dashed boxes. (C) BACTH analysis confirms the importance of residues Val29, Asn31 and Ser36 for self-interaction of the RapZ-NTD. β-Galactosidase activities of strains producing variants of the RapZ-NTD fused to both, the T25- and T18-fragments of CyaA are shown in the left panel. The right panel shows the RapZ tetramer structure, with the NTD:NTD interaction probed in this assay highlighted in the dashed boxes. (D) Mutation of residue Trp191 impairs the interaction between RapZ-NTD and CTD, but does not affect self-interaction of RapZ-CTD. β-Galactosidase activities of strains producing variants of the RapZ-NTD and RapZ-CTD fused to CyaA fragments are shown in the left panel. The right panel shows the RapZ tetramer structure, with the NTD:CTD interaction probed in this assay highlighted in the dashed boxes.

Figure 6.

Oligomerization of RapZ is required for activity and RNA binding. (A) Complementation assay demonstrating that correct multimerization is essential for activity of RapZ in vivo. Plasmids encoding RapZ-F181A, RapZ-D182A or wild-type RapZ were introduced into strain Z28, which lacks the endogenous rapZ gene and carries an ectopic glmS’-lacZ reporter fusion in the λattB site on the chromosome. Cells carrying the empty expression vector pBAD33 (‘empty plasmid’) served as negative control. Arabinose (Ara) was added to induce and glucose (Glc) to repress expression of the rapZ alleles from the P_Ara promoter. Cells were grown to exponential phase followed by assessment of β-galactosidase activity. (B) Total cell extracts from (A) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining to directly assess the levels of GlmS, which becomes visible as a discrete band in ΔrapZ strains (5).

Figure 7.

Assessment of the role of individual domains of RapZ in RNA binding. (A) RNA binding activity is associated with the RapZ-CTD but not with the NTD. Full-length Strep-RapZ, Strep-RapZ-CTD and Strep-RapZ-NTD were purified via StrepTactin affinity chromatography from strain Z903 lacking endogenous rapZ. Aliquots of the elution fractions (E) were analyzed by 15% SDS-PAGE/Coomassie staining (top) and in parallel used to extract RNA, which was subsequently analyzed by northern blotting using a probe specific for GlmY (bottom). The amounts of RNA loaded were adjusted to equal protein concentrations, as determined by the SDS-PAGE gel (top). In addition, the cell extracts (CE) of the various strains harvested prior to protein purification were analyzed alongside the elution fractions. Right: EMSA experiments using purified proteins and radio-labelled GlmY. (B) Wild-type Strep-RapZ-CTD or mutants V180G, F181A and D182A were purified by StrepTactin affinity chromatography from strain Z903 and analysed as in (A). Right: EMSA experiment showing that RapZ CTD harbouring the F181A mutation is unable to bind radiolabelled GlmY in vitro. (C) RapZ-CTD binds RNA targets with higher affinity than the full-length protein. Dissociation constants were calculated for full-length RapZ (1–284) or RapZ-CTD (154–284) by biolayer interferometry using an Octet Red 96 device. The measurements were performed by fitting the response from three independent experiments, and error bars represent standard deviations from the mean.

Figure 8.

The role of RapZ domains in the interaction with RNase E. (A) GlmZ (20 nM) and RNase E NTD were mixed together in a 2:6 molar ratio and cleavage was allowed to proceed for the prescribed time at 30°C in the presence and absence of full length RapZ or the RapZ-CTD alone. The products of the degradation assay of GlmZ FL by catalytically active RNase E NTD (1–529) are identical irrespective of whether the C-terminal domain of RapZ is absent or present, whereas the presence of full length RapZ alters the processing pattern of GlmZ. (B) EMSA assays to probe the interaction between RNA, RNase E catalytic domain and RapZ domains. GlmZ, RapZ, RapZ-CTD and RNase E catalytic domain were mixed in equimolar ratios and incubated for 30 min at 30°C before being run on a native polyacrylamide gel and stained for RNA with SYBR Gold. Complexes are highlighted with red boxes.

Figure 9.

A structure gallery of components involved in RapZ-mediated regulation of bacterial amino-sugar metabolism. Left: the RNase E catalytic domain tetramer (PDB code: 2BX2) with the four protomers coloured red, blue, green and yellow. Middle: the RapZ tetramer with protomers coloured red, blue, green and yellow. Right: a molecular model of the post-cleavage GlmZ RNA (generated by SimRNAweb). The purpose of this model is to give an indication of the relative sizes of the ternary complex components, rather than to suggest the three-dimensional structure adopted by GlmZ. All models are shown in the same relative scale.