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.

analysis of the underlined "red" and "green" peaks, indicating that the two proteins do not co-elute from the gel fitration column.

SUPPLEMENTARY MATERIALS AND METHODS
For construction of plasmids strain Xl1-blue was used. Site-directed mutagenesis was performed using the combined chain reaction (CCR) protocol (Bi & Stambrook, 1998). Briefly, 5'phosphorylated oligonucleotides carrying the desired mutations were incorporated by thermostable ampligase into PCR fragments during amplification. Two hybrid constructs created in this study encode fusions of full-length or truncated RapZ variants to T25 or T18 domains of B. pertussis adenylate cyclase. To construct plasmids which carry fusions of RapZ NTD (aa 1-152) to T25 or T18 domain, inserts were PCR-amplified from full-length rapZ using BG637/BG1222 primers, digested with XbaI and KpnI and cloned into pKT25 (creating pSD9) or pUT18C (creating pSD11) plasmids. Similarly, for constructs which encode RapZ CTD (aa 153-284) fused to T25 or T18 domain, PCR fragments were amplified from full-length rapZ utilizing BG1223/BG639 primers and inserted between XbaI/KpnI sites in pKT25 (pSD10) or pUT18C (pSD12) plasmids. RapZ NTD mutants harboring V29W and N31W exchanges were introduced by CCR using phosphorylated mutagenesis primers BG1396 (V29W, pSD53) and BG1397 (N31W, pSD54). Forward and reverse primers as well as subsequent cloning procedure were identical to those described for pSD9. Plasmid pSD57 is also isogenic to pSD9, but carries a L36S mutation in the rapZ gene. It was generated as described for plasmid pSD9, but plasmid "rapZ 1.1" was used as template for PCR. Plasmid "rapZ 1.1" was recovered in a genetic screen and will be described elsewhere. To establish V29W, N31W and L36S exchanges in RapZ NTD fused to T18, inserts were excised via XbaI/KpnI from pSD53, pSD54 and pSD57 and cloned into pUT18C thereby generating pSD64 (V29W), pSD65 (N31W) and pSD80 (L36S), respectively.
In order to generate complementation constructs expressing rapZ -F181A or rapZ-D182A from the arabinose-inducible P Ara promoter, CCRs were performed using outward primers BG1049/BG397 and the mutagenesis primers BG1367 and BG1213, respectively. The resulting CCR fragments were inserted between the SacI/XbaI sites on plasmid pBAD33 generating plasmids pBGG459 and pBGG436.
For construction of a plasmid encoding the Strep-tagged RapZ NTD, a fragment was amplified from full-length rapZ using BG1015/BG1358 primers, and ligated between the NheI/XbaI sites on plasmid pBGG237 thus creating pSD25. Using primers BG1340/BG397 plasmid pSD24 coding for Strep-RapZ CTD was created in a similar manner. Plasmids pSD98, pSD50, and pSD99 are isogenic to pSD24 but harbor mutations V180G, F181A and D182A, respectively. The rapZ-CTD fragments carrying mutations F181A (pSD50) and D182A (pSD99) were amplified using primers BG1340/BG397 and plasmids pBGG455 and pBGG432 as templates, respectively, and subsequently the PCR fragments were ligated with the NheI/XbaI-digested plasmid pBGG237.
Plasmid pSD98 is isogenic but carries mutation V180G in rapZ. It was obtained by ligation of a CCR fragment, which was generated using primers BG1340/BG397 and mutagenesis primer strep-rapZ (aa 1-152) under Ptac promoter in pBGG237 this work pSD30 as pSD10, but rapZ with F181A mutation this work pSD31 as pSD12, but rapZ with F181A mutation this work pSD32 as pSD10, but rapZ with D182A mutation this work pSD33 as pSD12, but rapZ with D182A mutation this work pSD46 as pBGG348, but rapZ with Y179A mutation this work pSD48 as pSD10, but rapZ with Y179A mutation this work pSD50 as pSD24, but rapZ with F181A mutation this work pSD52 as pSD10, but rapZ with W191A mutation this work pSD53 as pSD9, but rapZ with V29W mutation this work pSD54 as pSD9, but rapZ with N31W mutation this work pSD57 as pSD9, but rapZ with L36S mutation this work pSD60 as pSD12, but rapZ with Y179A mutation this work pSD64 as pSD11, but rapZ with V29W mutation this work pSD65 as pSD11, but rapZ with N31W mutation this work pSD80 as pSD11, but rapZ with L36S mutation this work pSD96 as pSD12, but rapZ with V180G mutation this work pSD97 as pSD10, but rapZ with V180G mutation this work pSD98 as pSD24, but rapZ with V180G mutation this work pSD99 as pSD24, but rapZ with D182A mutation this work pUT18C Plac cyaA-T18 (aa 225-399), MCS, bla, ori ColEI (Karimova et al., 1998)