SUMMARY
The global regulator Hfq facilitates the action of regulatory RNAs in post-transcription gene regulation in many Gram-negative bacteria. In Pseudomonas aeruginosa, Hfq, in conjunction with the catabolite repression protein Crc, was shown to form a complex that directly inhibits translation of target transcripts during carbon catabolite repression. Here, we describe and validate high-resolution cryo-EM structures of the cooperative assembly of Hfq and Crc bound to a translation initiation site. The core assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers can be recruited to form higher-order assemblies with demonstrated in vivo activity. The structures indicate a distinctive RNA conformation and a pattern of repeating motifs that confer regulatory function. This study not only reveals for the first time how Hfq cooperates with a partner protein to regulate translation but also provides a novel structural basis to explain how an RNA code can guide global regulators to interact cooperatively and regulate many different RNA targets.
INTRODUCTION
The control of gene expression in many bacteria is fine-tuned through intricate post-transcriptional networks mediated by the action of small regulatory RNAs (Wagner and Romby, 2015). Many of these regulatory molecules require the RNA chaperone Hfq, which protects the RNA against ribonucleases and facilitates their base-pairing interactions with cognate RNA targets (Vogel and Luisi, 2011). Hfq is a member of the Lsm/Sm family and shares with that group an ancient structural core that oligomerizes to form toroidal architectures exposing several RNA-binding surfaces. Crystallographic and biophysical data show that RNA recognition is mediated by distinct interactions with distal, proximal and rim faces (Schumacher et al., 2002; Link et al., 2009; Sauer et al., 2012; Panja et al., 2013), as well as revealing the role of the natively unstructured C-terminal tail in autoregulating RNA binding activities (Santiago-Frangos et al., 2016; 2017).
In the opportunistic pathogen Pseudomonas aeruginosa, Hfq acts as a pleiotropic regulator of metabolism (Sonnleitner and Bläsi, 2014), virulence (Sonnleitner et al., 2003; Fernandez et al., 2016; Pusic et al., 2016), quorum sensing (Sonnleitner et al., 2006; Yang et al., 2015) and stress responses (Lu et al., 2016). Many of these roles are likely facilitated through partner molecules, and numerous putative protein interactors of P. aeruginosa Hfq have been identified with functions in transcription, translation and mRNA decay (Van den Bossche et al., 2014). In the case of Hfq from Escherichia coli, the functionally important partners include RNA polymerase, ribosomal protein S1 (Sukhodolets and Garges, 2003), the endoribonuclease RNase E (Ikeda et al., 2011), polyA-polymerase, and the exoribonuclease polynucleotide phosphorylase (Mohanty et al., 2004; Bandyra et al., 2016). Most likely, these complexes are RNA mediated and affect the colocalisation of the transcriptional, translational and RNA decay machineries (Worrall et al., 2008; Resch et al., 2010; Večerek et al., 2010).
One P. aeruginosa protein that was found to co-purify with tagged Hfq is the Catabolite repression control protein, Crc (Van den Bossche et al., 2014; Moreno et al., 2015; Sonnleitner et al., 2018). Crc is involved in carbon catabolite repression (CCR) in Pseudomonas, a process that channels metabolism to use preferred carbon sources (such as succinate) until they are exhausted, whereupon alternative sources are used (Rojo, 2010). Crc strengthens binding of A-rich target transcripts to the distal side of Hfq (Sonnleitner et al., 2018). In this way, translational repression arises through binding of both Hfq and Crc to A-rich ribosome-binding sequences of mRNAs sensitive to catabolite repression, such as amiE mRNA, encoding aliphatic amidase (Sonnleitner et al., 2009; Sonnleitner and Bläsi, 2014). The translationally repressed mRNA, e.g. amiE, is then subjected to degradation, which might trigger disassembly of the Hfq/Crc/RNA complex (Sonnleitner and Bläsi, 2014). The non-coding RNA CrcZ, which increases in levels when the preferred carbon source is exhausted, was shown to sequester Hfq, and thereby to counteract Hfq/Crc mediated translational repression of mRNAs related to catabolism (Sonnleitner et al., 2009; Sonnleitner and Bläsi, 2014). CrcZ expression is under control of the alternative sigma factor RpoN and the two-component system CbrA/B (Sonnleitner et al., 2009). Although the signal responsible for CbrA/B activation remains unknown, it is thought to be related to the cellular energy status (Valentini et al., 2014).
In addition to carbon catabolite repression, Hfq and Crc link key metabolic and virulence processes in Pseudomonas species. The two proteins affect biofilm formation, motility (O’Toole et al., 2000, Huang et al., 2012, Zhang et al., 2012; Pusic et al., 2016), biosynthesis of the virulence factor pyocyanin (Sonnleitner et al., 2003; Huang et al., 2012), and they have been shown to affect antibiotic susceptibility (Linares et al., 2010; Heitzinger, 2016). Recent ChiP-seq studies indicate that Hfq and Crc have an even broader regulatory impact in Pseudomonas. It was shown that these regulators can work in concert to bind many nascent transcripts co-translationally, uncovering a large number of regulatory targets (Kambara et al., 2018).
To gain insight into how P. aeruginosa Hfq cooperates with Crc in translational repression of mRNAs, we determined the structure of the complex they form on the Hfq binding motif of the CCR-controlled amiE mRNA using cryo-electron microscopy (cryoEM). Our analyses revealed that the components form higher order assemblies and explain for the first time how a recurring structural motif can support the association of Hfq and RNA into cooperative ribonucleoprotein complexes that have key regulatory roles. We observe that the interactions supporting the quaternary structure are required for in vivo translational regulation. These findings expand the paradigm for in vivo action of Hfq through cooperation with the Crc helper protein and RNA to form effector assemblies.
RESULTS
An ensemble of Hfq/Crc/amiE6ARNRNA assemblies
For cryo-EM structural studies of the Hfq/Crc/RNA complex, purified recombinant Hfq and Crc proteins were mixed with an 18 nucleotide Hfq binding motif from the translation initiation region of the CCR-controlled amiE mRNA, which encodes aliphatic amidase (hereafter, amiE6ARN). This binding motif comprises of 6 repeats of an A-R-N pattern preferred by the distal face of Hfq. The purified sample of Hfq/Crc/amiE6ARN, after mild chemical crosslinking, yielded well defined single particles on graphene oxide in thin, vitreous ice. Analysis of the reference free 2D class averages and subsequent 3D classification indicated three principal types of complexes corresponding to different stoichiometries of Hfq (hexamer):Crc:amiE6ARN with compositions 2:2:2, 2:3:2 and 2:4:2 (Figure 1). These higher order assemblies are in agreement with recently observed SEC-MALS and mass spectrometry results which excluded a simple 1:1:1 assembly (Sonnleitner et al., 2018). The maps for the complexes are estimated to be 3.12 Å, 3.27 Å and 3.27 Å in resolution, respectively, based on gold-standard Fourier shell correlations (Figure 1 – figure supplement 1). The distribution of the complexes corresponds to roughly 40%, 33% and 26% for the 2:2:2, 2:3:2 and 2:4:2 complexes (Figure 1). The individual crystal structures of Hfq and Crc dock well into the cryoEM densities (Figure 1 and Figure 1 – figure supplement 2), and aside from side chain rotations there are few other structural changes of the components (Figure 1 – figure supplement 2). CryoEM analyses of samples that had not been treated by crosslinking show that the quaternary structure was not affected by the treatment (Table S1).
In the core complex (2:2:2), the two Hfq hexamers sandwich the RNA and Crc components (Figure 2A). Each Hfq interacts with one amiE6ARN RNA and two Crc molecules, forming an assembly with C2 symmetry. The molecular twofold axis passes through the centre of the two Crc molecules, and the same Crc-to-Crc interface is observed in the crystal structure of the isolated Crc dimer (generated through crystallographic symmetry) (Milojevic et al., 2013). As anticipated, the dominating protein/RNA interaction is made by the distal face of Hfq, forming an interface area of ∼2270 Å2. The two Crc molecules interact with RNA residues exposed on the surface of Hfq, and both Crc molecules contact the Hfq-rim on the distal side (Figure 2A).
The Crc forms antiparallel dimers in the 2:2:2-complex, so there are two modes of interaction with the amiE6ARN RNA. Each binding mode is used once at either of the interfaces with Hfq/RNA (Figure 2A). In each Crc monomer, Arg140 η1-NH2 and Arg141 ε-NH and η1-NH2 interact with the phosphodiester backbone of amiE6ARN. Arg140 and Arg196 form a sandwich with the purine-base of the A3 nucleotide at an entry/exit site of amiE6ARN RNA. The Arg196 ε-NH and η1-NH2 groups form hydrogen bonds with the U6-amiE6ARN backbone and the U6 O2 group forms a hydrogen bond with the Met156 amide. In the second mode of interaction, Lys155 ζ-NH2 makes a hydrogen bond with the OP2-group of C9 and the ribose hydroxyl group. Additional hydrogen bonds are formed between Trp161 ε1-NH and Arg162 η1/2-NH2 and the phosphate backbone of amiE6ARN (Figure 2A). These highly organised interactions illustrate how the bases of amiE6ARN as presented by Hfq constitute a molecular interface for the RNA-mediated interactions between Hfq and Crc.
The quaternary organisation of the 2:2:2 complex forms a core unit that is also present in the 2:3:2 and 2:4:2 complexes. In that common core, the interaction of the Crc with the RNA leaves approximately half of the accessible surface of the nucleic acid exposed. For the 2:3:2 and 2:4:2 complexes, additional Crc units are recruited through interactions with the exposed portion of the RNA (Figure 3A). As such, the C2 symmetry is broken by the third Crc molecule in the 2:3:2 complex (Figure 1). Interestingly, recruitment of a fourth Crc monomer to the complex restores the C2 symmetry, preserving the symmetry axis from the core complex, but with a conformationally different Crc dimer interface between Crc molecules 3 and 4 (Figure 3A). The two additional Crc monomers have small surface-area contacts with the rest of the complex and are likely to be comparatively mobile, which may account for the stronger variation in resolution for the 2:3:2 and 2:4:2 maps compared to the rather rigid 2:2:2 core assembly (Figure 1-figure supplement 1).
Function, origins and validation of subunit cooperativity in the 2:2:2 complex
Hfq binds the amiE6ARN RNA avidly with a dissociation constant in the nanomolar range, but in contrast Crc has no intrinsic RNA-binding activity (Milojevic et al., 2013). In the presence of Crc, the off-rate for Hfq on amiE6ARN decreases (Sonnleitner et al., 2018), which indicates a cooperation of the components in binding RNA. The complexes revealed here show that Crc forms small contact surfaces to the RNA, to Hfq, and to itself as a homodimer; these small areas work together to give an assembly that is most likely stabilised through chelate cooperativity. Notably, there is a striking absence of any lower order assemblies in the cryo EM micrographs. The 2:2:2 complex is therefore likely to be the minimal complex formed when all components are present and must be constructed in an ‘all or nothing’ manner, somewhat like a binary switch.
The dimer interface of the Crc pair is the largest protein-protein interface in the 2:2:2-complex and has a buried area of 766 Å2, which typically corresponds to a moderate intermolecular affinity. The key dimerization interface is maintained by salt bridges between Arg229-Arg230 of one Crc monomer and Glu142 of the second Crc monomer, which is further stabilised by pi-stacking of the Phe231-Phe231 rings at the point of symmetry (Figure 2A). The phenylalanine residues are in turn stabilised by stacking interactions with Trp255 of the same Crc monomer (not shown). Two additional polar contacts are formed between Arg137 and the Asn184 carbonyl group of two pairs of helices in the Crc dimer, forming a smaller secondary interface (Figure 2A).
The observed interactions shown for the 2:2:2 Hfq:Crc:RNA complex (Figure 2A) are consistent with genetic, biochemical and biophysical data, which revealed intermolecular interactions between Crc protomers, interactions between Crc and RNA as well as a few interactions between Crc and Hfq. These data also showed that formation of the Hfq/Crc/RNA complex requires binding of the RNA on the distal face of Hfq (Sonnleitner et al., 2018). To verify selected interactions between the Crc protomers and Crc and RNA, we explored the effects of mutations on translational repression of an amiE::lacZ reporter gene by Hfq and Crc (Sonnleitner et al., 2018), and on the capacity of Hfq and Crc to co-immunoprecipitate in the presence of amiE6ARN RNA. First, we asked whether R140 (CrcR140) is required for the interaction of the protein with the RNA (Figure 2A, bottom left inset). As shown in Figure 2B, the CrcR140E mutant was deficient in repression of the amiE::lacZ reporter gene, similarly as observed in the crc deletion strain. Moreover, CrcR140E did not co-immunoprecipitate with Hfq in the presence of amiE6ARN RNA (Figure 2 – figure supplement 1), strongly indicating that the interaction between CrcR140 and RNA is pivotal for Hfq:Crc:RNA complex formation.
Next, we focused on the possible role of the salt bridges between the E142 and R229/R230 ‘triangle’ (Figure 2A, top left inset) for the Crc-Crc interaction. The single mutant proteins CrcR229E and CrcR230E did not affect translational repression of amiE::lacZ, whereas the function of the CrcE142R variant was diminished (Figure 2B), indicating that E142 can form salt bridges with either R229 or R230. The de-repression of amiE:lacZ observed with the CrcE142R variant was partially compensated by the double mutant proteins CrcE142R, R229E and CrcE142R, R230E. In addition, the CrcE142R and CrcR230E variants were impaired in Hfq:Crc:RNA complex formation as shown by the co-immunoprecipitation assay (Figure 2 – figure supplement 1A). Strikingly, the compensatory changes present in the triple mutant protein CrcE142R, R229E, R230E almost fully restored translational repression of the amiE::lacZ reporter gene. As the respective Crc variant proteins were produced at comparable levels (Figure 2 – figure supplement 1B), these mutational studies support the in vivo role for the interactions of the Crc protomers observed in the cryo-EM models.
Function, origins and validation of subunit cooperativity in the 2:4:2 complex
The protomer interactions of the 2:2:2 assembly are highly interdependent, and once the core complex is generated it can recruit additional Crc molecules, forming the 2:3:2 and 2:4:2 complexes. In the 2:4:2 complex, a second type of Crc dimer seems to assemble with a smaller buried surface (Figure 3A). Such additional dimers can only form when an intact 2:2:2 core complex is present, as they are not observed in the core complex itself nor in solution or through crystallographic symmetry (Milojevic et al., 2013). Notably, the additional dimer is a more ‘open’ conformation of the crystallographic Crc dimer in the core, which is further supported by normal mode analysis (data not shown). The same key Crc dimer interface is occupied but seems to serve as a dynamic hinge, whereas the secondary, smaller, dimer interface between the Crc helices is absent to allow the new Crc dimer to adopt an ‘open’ conformation. Arg230 is reorganised by Glu193 in the same protomer to self-interact with the corresponding Arg230 in the partner Crc, rather than with Glu142 (Supplementary movie 1). Additional hydrogen bonds are formed between Arg233 and Glu193, whereas Arg229 is no longer part of the dimer interface (Figure 3A). Both Arg230 and Glu193 seem to play pivotal roles in providing the structural freedom to form a dynamic hinge (Figure 3C).
Only the Arg233-Glu193 interaction is unique for the 2:4:2 assembly and was assessed in vivo. Strikingly, CrcE193R fully abrogated repression of the amiE:LacZ reporter gene (Figure 3B). The model predicts that the deleterious CrcE193R mutation can be compensated by the substitution of CrcR230E to re-establish the interaction. This pair does indeed behave as predicted, further confirming the in vivo importance of the 2:4:2 assembly during CCR (Figure 3B). By reorganising the extra Crc molecules 3 and 4 that bid the 2:2:2 core (Figure 3A), the alternative Crc dimer is able to utilise one of two basic patches on its surface when engaging amiE6ARN without causing steric hindrance to the already bound crystallographic dimer.
In addition to Crc Arg140 and Arg141, Crc K139 ζ-NH2 makes a hydrogen bond with the OP2- group of A12, Arg138 η1-NH2 interacts with the ribose hydroxyl group of C9 and K135 ζ-NH2 forms a hydrogen bond with the A11 OP2. Finally, the O2 of cytosine C12 engages in a hydrogen bond with the backbone amino group of Arg140. Direct interactions between the reorganised Crc dimer and Hfq are limited to the same Crc β-strand and exposed loop of a sole Hfq monomer, as in the core complex. Due to the open conformation of the alternative Crc dimer, the Hfq Thr49 hydroxyl group now forms a hydrogen bond with the Ala 78 amide group (Figure 3A).
Interestingly, a basic half-channel is formed over the core dimer interface, with additional basic patches spread over the RNA binding surface of the Crc dimer (Figure 3D). Speculatively, longer RNA species could travel though the surface exposed half-channel and interconnect all components of the core complex into a highly organised assembly on this target RNA.
A specialised and recurring RNA conformation in Hfq-mediated regulation
Link et al. (2009) described the crystal structure of E. coli Hfq bound to a polyriboadenylate 18-mer and observed that the RNA encircled the distal face of the Hfq hexamer via a repetitive tripartite binding scheme. Each base triplet is partially embedded between adjacent Hfq monomers and is mostly surface exposed, folding into a ‘crown-like’ conformation. We observe striking similarities with the fold of the authentic amiE6ARN species on the distal side of the P. aeruginosa Hfq hexamer (Figure 4). Notably, the cryoEM maps were calculated without any reference to the Link et al. (2009) structure. The agreement between the co-crystal structure of the homologous complex and the entirely independently derived cryoEM based model is a strong validation of both experimental procedures, X-ray crystallography and cryoEM. A recent study proposed an RNA-RNA stacking interface between two RNA species presented by Hfq, supported by crystal structures and biophysical analysis in solution (Schulz et al., 2017). Although all components necessary for such interaction are present in our reaction mixture, we do not observe such dimeric species by cryo-EM or in solution when Crc is present.
Like its E. coli homologue, Pseudomonas Hfq contains 6 tripartite binding pockets on the distal side, capable of binding a total of 18 nucleotides. Each of the six RNA triplets of the amiE6ARN RNA fits into an inter-subunit cleft in Hfq (Figure 4). The specific, star-shaped RNA fold is guided by six positively charged protuberances on the distal face of Hfq, with the phosphate backbone circularly weaving in between these, seemingly to minimise steric hindrance while maximizing surface interactions (Figure 4). As described by Link et al. (2009), each pocket consists of an adenosine specificity site (A), a purine nucleotide specificity site (R), and a presumed RNA entrance/exit site (E) which is non-discriminatory. Hfq thus has a structural preference for (ARN)n RNA stretches on its distal side, where N is any nucleotide. The adenosine specificity (A) sites are organised identically to the corresponding A sites in E. coli Hfq, forming hydrogen bonds between the peptide backbone and carboxyl-groups of Gln33 and the N6,7 atoms of the adenosine base, and a polar interaction between Gln52 (Nε) and the N1 atom of the adenosine base. The peptide backbone amide of residue Lys31 interacts with the 5’ phosphate group of adenine. Finally, the adenine base is stacked against the side chain of Leu32 (Figure 4). The purine (R) specificity site is defined by two neighbouring monomers, where the side chains from Tyr25 and from Leu26’, Ile30’ and Leu32’ (where the prime denotes residues from a neighbouring subunit) contact the nucleotide aromatic base. In amiE6ARN, one R-site is populated by a guanine, forming a hydrogen bond between the Nε of Gln52’ and the guanine exocyclic O6 (Figure 4). Just like in the E. coli Hfq/polyA18 structure (Link et al., 2009), Gln52’ forms a physical link between the A and R sites. Previous structures were obtained from polyA RNA, whereas the structures presented here were solved with the authentic amiE Hfq recognition site. Interestingly, Thr61 Oγ forms a double hydrogen bond with the N1 and the exocyclic N2 from the guanine base, which was not seen previously (Link et al., 2009) as all R-sites were occupied by adenine residues (Figure 4).
Discussion
Many functional studies have highlighted how global posttranscriptional regulators cooperate with each other and their RNA targets to control the fate of transcripts with high specificity. A major gap in our current understanding has been the lack of high resolution structural data of these highly coordinated cellular processes. Here we report the first atomic model of Hfq interacting with a translational initiation region (amiE6ARN) and a partner protein to form a multi-component assembly that mediates translational control (Kambara et al., 2018; Sonnleitner et al., 2018). The RNA is a recurring A-rich fragment of amiE that occupies almost entirely the distal surface of Hfq, weaving in between basic, surface exposed islands. There are striking similarities to the structure of the polyA18 complex with E. coli Hfq reported by Link et al. (2009), whose structure greatly added to the understanding of RNA binding and chaperone mechanisms, and hinted at how the distinct polyA RNA interaction might enable Hfq-mediated regulation. The polyA/Hfq structure revealed rules for recognition of motifs of the type A-R-N, where R is purine and N is any base. The P. aeruginosa Hfq interaction with amiE6ARN follows the same rules. The A-R-N repeat occurs in many RNAs, and it has been proposed that the exposed bases could mediate RNA to RNA interactions (Schulz et al., 2017). It is also a recurring motif in the nascent transcripts that are associated with Hfq and Crc in Pseudomonas (Kambara et al., 2018). We observe that the exposed bases (entry/exit site) and RNA backbone in the Hfq/amiE6ARN complex are available for interactions with Crc to form a cooperative assembly that efficiently mediates catabolite repression in vivo when the preferred carbon source is available (Figure 5).
Previous studies have shown that both Hfq and Crc are required for tight translational repression of mRNAs, which are subject to carbon catabolite repression (CCR) (Sonnleitner and Bläsi, 2014; Moreno et al., 2015). The presence of Crc did not significantly enhance the affinity of Hfq for amiE6ARN RNA (Sonnleitner et al., 2018). However, the simultaneous interactions of Crc with both binding partners resulted in an Hfq/Crc/RNA assembly with increased stability when compared with the Hfq/RNA complex alone (Sonnleitner et al., 2018). In light of our structural studies, the enhancing effect of Crc in Hfq-mediated translational repression of target mRNAs during CCR (Sonnleitner and Bläsi, 2014; Moreno et al., 2015) can be readily explained by the interactions of Crc with both binding partners. It also accounts for the observed decrease in the off-rate on the RNA substrate (Sonnleitner et al., 2018). It is conceivable that full repression is only achieved when amiE6ARN is masked entirely in the 2:4:2 complex, which is supported by our in vivo studies.
The question arises why a higher order assembly such as the 2:2:2 core is formed and not a simpler complex. The structural data indicate that the dimerization of Crc provides the key step for formation of the 2:2:2 complex, because it will pre-organise a copy of the surface that interacts with the Hfq/RNA so that a second Hfq/RNA complex can be recruited. Thus, all components are necessary to form the complex so that there is no formation of lower order ‘sub assemblies’. The structural data are consistent with Crc having no capacity for RNA binding by itself (Milojevic et al., 2013). The Hfq/Crc/RNA complex is thus assembled in a checklist-like manner through numerous small contacting surfaces and when the RNA target is presented by Hfq in a specific, well-defined configuration. In this way, the components interact mutually through chelate cooperative effects. Most likely the 2:2:2 core forms first, then the other Crc components are recruited.
We envisage that the 2:2:2 core and higher order assemblies might interact with other longer RNAs. The higher order assembly could capture two of such mRNA substrates (Figure 5), but chelate effects might instead induce formation of the complex on a single mRNA target. In that scenario, a portion of the mRNA would thread through the central basic half channel as depicted in Figure 3D. Under conditions of catabolite repression regulation, pull-down assays showed that Hfq and Crc form a co-complex in the presence of the 426nt long CrcZ RNA (Moreno et al., 2015; Sonnleitner et al., 2018). In the presence of less preferred carbon sources, the expression levels of CrcZ RNA increase (Sonnleitner et al., 2009) and CrcZ functions as an antagonist in Hfq/Crc mediated translational repression of catabolic genes. The CrcZ RNA has multiple ARN triplets that could be sites for Hfq/Crc interaction (Sonnleitner and Bläsi, 2014) that could sequester multiple Hfq/Crc proteins (Figure 5). Thus, under conditions where CCR is relieved, CrcZ RNA would serve as a sponge for Hfq/Crc to prevent repression of genes encoding proteins required for utilizing the less preferred carbon sources (Figure 5). How the CrcZ RNA is displaced from Hfq/Crc remains unknown. However, the assemblies are likely to be dynamic and the displacement process might resemble that proposed for the step-wise exchange of sRNAs on Hfq (Fender et al., 2010). Recent findings show that the regulatory spectrum of Hfq and Crc is much broader than initially expected. Hfq was found to bind more than 600 nascent transcripts co-transcriptionally often in concert with Crc (Kambara et al., 2018). These findings indicate that Hfq and Crc together regulate gene expression post-transcriptionally beyond just catabolite repression.
Understanding how gene expression is regulated post-transcriptionally in pathogens such as P. aeruginosa may provide potential targets for novel drug design. Hfq and Crc are involved in key metabolic and virulence processes in Pseudomonas species (O’Toole et al. 2000; Sonnleitner et al., 2003; Sonnleitner et al., 2006; Linares et al., 2010; Huang et al., 2012; Zhang et al. 2012; Zhang et al., 2013; Sonnleitner and Bläsi, 2014; Pusic et al., 2016). Disrupting the interface of the core assembly of the Hfq/Crc complex might be one strategy to counter, among other, metabolic regulation and consequently its downstream processes that impact on virulence during infection. A recent study showed how overproduction of the aliphatic amidase AmiE strongly reduced biofilm formation and almost fully attenuated virulence in, amongst others, a mouse model of acute lung infection (Clamens et al., 2017). Novel drugs that specifically counteract Hfq:Crc:amiE assembly formation and prevent repression of AmiE production could induce the phenotype described by Clamens et al (2017). The high resolution structures presented here provide a starting point for novel strategies to interfere with e.g. carbon regulation in a pathogenic bacterium for therapeutic intervention of threatening infections.
MATERIALS AND METHODS
Protein synthesis, purification and complex formation
P. aeruginosa Hfq and Crc were produced in E. coli and purified as described by Sonnleitner et al. (2018). The synthetic 18-mer amiE6ARN RNA (5’-AAAAAUAACAACAAGAGG-3’) used in these studies consists of six tripartite binding motifs (Sonnleitner and Bläsi, 2014). The Hfq/ Crc/ RNA complex was prepared by first heating the amiE6ARN RNA at 95°C for 5 minutes followed by 50°C for 10 minutes and 37°C for 10 minutes. The RNA was then incubated with the Hfq hexamer at a 1:1 molar ratio on ice for 20 minutes to form a binary complex, then an equal molar ratio of Crc was added. The mixture was incubated on ice for 30 minutes prior to fractionation by size exclusion chromatography using a Superdex 200 column equilibrated in running buffer composed of 20 mM HEPES, pH 7.9, 10 mM KCl, 40 mM NaCl, 1 mM MgCl2, and 2 mM TCEP (tris(2- carboxyethyl)phosphine). The peak fractions were buffer exchanged into 20 mM HEPES, pH 7.9, 10 mM KCl, 40 mM NaCl, 5 mM MgCl2. Samples used for cross-linking were incubated with bis(sulfosuccinimidyl)suberate (BS3) at 150 μM for 30 minutes on ice, followed by quenching at 37.5 mM Tris-HCl pH 8.0.
CryoEM specimen preparation and data acquisition
Graphene oxide grids are prepared as described by Pantelic et al. (2010). Briefly, 2 mg/ml of graphene oxide solution in water (Aldrich) was diluted ten times in water. After removing aggregation by spinning for 30 seconds at 300 rcf, 2 μl of graphene oxide solution was loaded on freshly glow discharged quantifoil Au-grids (R1.2/1.3, 300 mesh). Glow discharge was performed prior to graphene oxide coating at 45 mA for 60 second with an Edward Sputter Coater S150B at 0.2m Bar at 0.75 KV. After the graphene oxide had been adsorbed for 1 minute, the grids were washed 3 times with 20 μl water, then air-dried for 1 hour at room temperature prior to sample application. Specimens for cryoEM analysis were prepared by applying 2 μl of a 0.65 μM solution of the Hfq/Crc/RNA complex to the Quantifoil Au grids freshly coated with graphene oxide. After an adsorption time of 60s, the grids were blotted for 10 seconds at a blot force of 5, then plunge frozen into liquid ethane using a Vitrobot (FEI). Images were recorded on a Krios G2, Falcon III direct electron detector at 300 kV operating in counting mode (Supplementary Table 3).
Movie processing, single particle analysis, 3D reconstruction and refinement
Whole frame motion correction was performed on movies with motioncorr2 with dose weighting followed by CTF estimation using gctf (Zhang, 2016; Zheng et al., 2017). RELION-2.1 was used for data processing (Scheres, 2012). Final resolution estimates were calculated after the application of a soft binary mask and phase randomisation and determined based on the gold standard FSC=0.143 criterion (Scheres and Chen, 2012; Chen et al., 2013).
For the BS3 treated complex, after manually picking 3159 particles and using suitable references for autopicking, 482426 particles were used for early classifications. After three rounds of rejecting particles by 2D classification, 215774 particles were used for initial model generation and 3D classification. An initial model was generated using an SGD algorithm based on a small subset of particles with diverse orientations (Punjani et al., 2017). During 3D classification, three different complexes were resolved after 25 iterations with an angular sampling of 7.5°: 2Hfq:2Crc:2amiE6ARN (2:2:2), 2Hfq:3Crc:2amiE6ARN (2:3:2) and 2Hfq:4Crc:2amiE6ARN (2:4:2). To properly separate, validate and refine the 3 classes, the same 3D classification was rerun with the new 2:3:2 model as reference model, lowpass filtered to 20 Å resolution. C2 symmetry was observed and imposed for the 2:2:2 and 2:4:2 complexes. Each of the classes was then refined to sub-3.5 Å resolution, followed by per-particle frame alignment for movement correction and per-frame damage weighting. The resulting ‘polished’ particles were subjected to a final refinement round with solvent flattening. All reference models were lowpass filtered to 60 Å prior to refinement. The dominant class (2:2:2) had a resolution of 3.12 Å. Local resolution calculations were done with the relion local resolution estimator (Supplementary Figures 1 and 2A, Supplementary Table 1).
Crystal structures for P. aeruginosa Crc (PDB code 1U1S) and Hfq (PDB code 4JG3) were manually docked into the EM density map as rigid bodies in Chimera (Pettersen et al., 2004). The RNA 18-mers were manually built into the density using Coot (Emsley et al., 2010). Refmac5 and Phenix real-space refinement with global energy minimization, NCS-restraints, group B-factor and geometry restraints were used to iteratively refine the multi-subunit complexes at high resolution, followed by manual corrections for Ramachandran and geometric outliers in Coot (Supplementary Table 1) (Emsley et al., 2010; Murshudov et al., 2011; Afonine et al., 2012). Model quality was evaluated with Procheck in CCP4 and MolProbity (Williams et al., 2018). In silico 2 Å maps were generated from the atomic models and FSC validation against the experimental maps was performed with the EMDB Fourier shell correlation server (EMBL-EBI) (Figure 1 – figure supplement 2 B).
Bacterial strains and plasmids
The strains, plasmids and oligonucleotides used in this study are listed in Supplementary Tables S2 and S3.
Construction of plasmids encoding Crc variant proteins for in vivo translational repression assay
To test the proficiency of Crc mutant proteins to co-repress translation of a translational amiE:lacZ reporter gene, derivatives of plasmid pME4510crcFlag (Supplementary Table S2) were constructed by means of Quick change site directed mutagenesis (Agilent Technologies). Plasmid pME4510crcFlag was used together with the corresponding mutagenic oligonucleotide pairs (Supplementary Table S3). The parental plasmid templates were digested with DpnI and the mutated nicked circular strands were transformed into E. coli XL1-Blue, generating plasmids pME4510crc(R140E)Flag, pME4510crc(E142R)Flag, pME4510crc(R229E)Flag, pME4510crc(E193R)Flag, pME4510crc(R230E)Flag, pME4510crc(E142R, R229E)Flag, pME4510crc(E193R, R230E)Flag, pME4510crc(E142R, R230E)Flag and pME4510crc(E142R, R229E, R230E)Flag.
In vivo translational repression of an amiE::lacZ reporter gene in the presence of Crc variants
The ability of the Crc mutant proteins to repress translation of an amiE::lacZ reporter gene was tested in a PAO1 crc deletion strain bearing plasmids encoding the wt protein or the respective protein variants (Supplementary Table S2) as described by Sonnleitner et al. (2018). The β-galactosidase activities were determined as described (Miller, 1972). The β-galactosidase units in the different experiments were derived from two independent experiments.
Construction of plasmids employed for the production of selected Crc mutant proteins
The R140E, E142R, R230E single aa exchanges in Crc were obtained by using the QuickChange site-directed mutagenesis protocol (Agilent Technologies). The plasmid pETM14lic-His6Crc (Supplementary Table S2) was used together with the corresponding mutagenic oligonucleotide pairs (Supplementary Table S3). The entire plasmids were amplified with Pfu DNA polymerase (Thermo Scientific). The parental plasmid templates were digested with DpnI and the mutated nicked circular strands were transformed into E. coli XL1-Blue, generating plasmids pETM14lic-His6CrcR140E, pETM14lic-His6CrcE142R and pETM14lic-His6CrcR230E
Purification of Crc and Crc variants
The Crc protein and the Crc variants CrcR140E, CrcE142R and CrcR230E were purified from E. coli strain BL21(DE3) harboring either plasmid pETM14lic-His Crc or the respective derivatives using Ni-affinity chromatography, followed by removal of the His6-tag with GST-HRV14-3C ‘‘PreScission’’ protease as described by Milojevic et al. (2013).
In vitro co-IP studies
The co-IP studies in the presence of 40 pmol of Hfq-hexamer, 120 pmol of Crc protein or of the respective Crc mutant proteins and 40 pmol amiE6ARN RNA were performed as described (Sonnleitner et al., 2018).
Western blot analyses
Equal amounts of proteins were separated on 12% SDS-polyacrylamide gels, and then electro-blotted onto a nitrocellulose membrane. The blots were blocked with 5% dry milk in TBS buffer, and probed with rabbit anti-Hfq (Pineda) and rabbit anti-Crc (Pineda) antibodies, respectively. Immuno-detection of ribosomal protein S1 served as a loading control. The antibody-antigen complexes were visualized with alkaline-phosphatase conjugated secondary antibodies (Sigma) using the chromogenic substrates nitro blue tetrazolium chloride (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP).
Acknowledgements
The coordinates and cryoEM maps have been deposited in the PDB and the EMBD. BFL, XYP and TD are supported by the Welcome Trust (200873/Z/16/Z). TD is also supported by an AstraZeneca Studentship. U.B. and E.S. are supported by the Austrian Science Fund (FWF) (www.fwf.ac.at/en) [P28711-B22]. We thank our colleagues Jamie Blaza, Dima Chirgadze, Jiri Sponer, Miroslav Kreply, Kasia Bandyra, Steven Hardwick, Sjors Scheres, Joerg Vogel, Armin Resch and Nguyen Thi Bach Hue for advice, helpful discussions and support. For access and help at facilities, we thank Giuseppe Cannon and staff at the MRC-LMB EM Facility and Kasim Sader at Thermo Fisher Scientific Pharma CryoEM Facility, Nanoscience Centre of University of Cambridge.