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.

Small RNA ‐binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli

“…This observation suggested that binding of GlmY* by RapZ is controlled by GlcN6P, which we investigated using EMSA. As observed in previous studies (Göpel et al, 2013;Gonzalez et al, 2017), radiolabeled GlmY* was readily bound when incubated with increasing concentrations of RapZ (Fig 6D, left panel, lanes 1-4). Interestingly, GlmY* remained unbound when 7.5 mM GlcN6P was included in the assay (Fig 6D, left panel, lanes 5-7).…”

“…The RapZ‐CTD binds GlmY and GlcN6P on its own (Figs C and D). In agreement, the CTD contains a pocket potentially suited to accommodate a metabolite, which is in close proximity to the RBD or could be even part of it (Gonzalez et al , ). As the RapZ‐CTD responds on its own to GlcN6P by releasing GlmY (Fig D), GlcN6P and the sRNA may compete for access to the RapZ‐CTD.…”

“…Although the RapZ-CTD retains some interaction, this residual binding is not sufficient to activate glmY expression as shown by a complementation assay using low copy plasmids encoding the RapZ variants ( Fig EV1B). Moreover, introduction of an Asp182Ala substitution into full-length RapZ abrogating self-interaction of the CTD (Gonzalez et al, 2017), concomitantly abolishes interaction with QseE as well as QseF ( Fig EV1A). Apparently, both domains of RapZ contribute to binding and activation of QseE/QseF and proper oligomerization of RapZ is a prerequisite for interaction.…”

“…Cleavage depends on the “adaptor” function of RapZ, which binds GlmZ at its central stem loop and recruits RNase E by interaction with its catalytic domain (Göpel et al , , ). RapZ forms a swapped dimer of dimers, an assembly required for activity (Gonzalez et al , ). Possibly, RapZ and the likewise tetrameric RNase E catalytic domain sandwich GlmZ for cleavage in an encounter complex.…”

“…The RapZ protomer consists of two globular domains, of which the C‐terminal domain (CTD) can dimerize and bind RNA on its own. The RBD is apparently formed by the surface‐exposed 19 residues long C‐terminal tail enriched in positive charges and SR motifs (Göpel et al , ; Gonzalez et al , ), features also present in emerging non‐typical eukaryotic RBDs (Hentze et al , ). The RapZ‐CTD exhibits structural homology to 6‐phosphofructokinase (Gonzalez et al , ), re‐emphasizing a recently recognized relationship between metabolic enzymes and RNA‐binding activity (Hentze et al , ).…”