Elke Duchardt-Ferner

Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch.

Riboswitches are highly structured RNA motifs with gene regulatory activity located in the untranslated regions of mRNAs. They either modulate transcription termination or translation initiation through conformational changes triggered by direct interactions with small metabolite ligands. Many naturally occurring riboswitches are large and structurally very complex. In contrast, synthetic riboswitches—tailored gene regulatory elements for synthetic biology applications—are based on small in vitro selected RNA aptamers. Yet, despite a ligand affinity and specificity comparable to their natural counterparts only a few in vitro selected aptamers are regulatory active in vivo. Recently, Suess et al. engineered a riboswitch for the aminoglycoside antibiotic neomycin B by subjecting an in vitro SELEX-pool to an in vivo screening for gene regulatory activity in a yeastbased reporter gene assay. The resulting neomycin B and ribostamycin (Figure 1a) responsive RNA-element (N1) contains only 27 nucleotides in a bulged hairpin secondary structure (Figure 1b)—the smallest riboswitch functional in vivo identified to date. In sequence and secondary structure, N1 differs completely from an in vitro selected but regulatory inactive RNA-aptamer for the same ligand (R23). Instead it partially resembles the ribosomal A-site, the natural target for aminoglycoside antibiotics (Figure 1b). The NMR spectroscopic analysis of the N1 riboswitch complexed with ribostamycin identifies structural determinants for its regulatory activity and suggests a ligand binding mechanism based on conformational capture. Our results provide insights into the modularity of ligand binding sites in RNA and highlight structural and dynamic features N1 shares with the larger naturally occurring riboswitches as well as with other regulatory active aptamers. This knowledge may guide the future design of novel synthetic riboswitches for targeted in vivo applications. Structure of the N1–ligand complex—the “OFF”-state of the riboswitch: N1 represses gene expression upon binding to either neomycin B or the closely related but smaller ribostamycin. NMR spectra of N1 bound to either ligand (Supporting Information Figure S1) indicate that both complexes are formed with similarly high affinity and display a high degree of structural similarity suggesting that the contribution of ring IV of neomycin to the interaction is negligible. Thus, we determined the structure of the N1–ribostamycin complex, because of its superior spectral resolution for the ligand resonances, by NMR spectroscopy (see Table 1). Chemical shift assignments and coordinates have been deposited (BMRB code: 16609, pdb-code: 2kxm). The structure of ribostamycin-bound N1 consists of a continuous helical stem with canonical stacking interactions between the G5:C23 and the G9:C22 base pair despite the presence of a flexible three-nucleotide bulge (C6–U8) and a compactly folded apical hexaloop organized around a U-turn motif (U14–A16) closed by the U13:U18 base pair (Figure 1c–e). Ribostamycin rings I and II are sandwiched between the N1 major groove, in the region from G5:C23 to U13:U18 and A17 protruding from the apical loop (Figure 2). Ring III is located close to the backbone of the 3’-strand (U18 to G20). Simultaneous contacts of the ligand with the G5:C23 base pair below and G9:C22 above the bulge (Figure 2b) clamp together the lower and upper helical stem and thus enforce the uninterrupted coaxial helical stacking across the flexible C6–U8 internal bulge. The bulge itself is not interacting with the ligand. A detailed structural description of the N1– ribostamycin complex is given in the Supporting Information. A comparison of the N1–ribostamycin complex with other aminoglycoside binding RNAs reveals partial similarities to known aminoglycoside binding sub-motifs: The helical stem centered at the U10:U21 base pair is similar to the ribosomal [*] Dr. E. Duchardt-Ferner, S. R. Schmidtke, Prof. Dr. J. W hnert Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany) Fax: (+49)69-798-29527 E-mail: woehnert@bio.uni-frankfurt.de

Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B

To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5′ handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.

Structural and functional analysis of the archaeal endonuclease Nob1

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.

Influence of Heparin Mimetics on Assembly of the FGF-FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

NMR resonance assignments of an engineered neomycin-sensing riboswitch RNA bound to ribostamycin and tobramycin

The neomycin-sensing riboswitch is an engineered riboswitch developed to regulate gene expression in vivo in the lower eukaryote Saccharomyces cerevisiae upon binding to neomycin B. With a size of only 27nt it is the smallest functional riboswitch element identified so far. It binds not only neomycin B but also related aminoglycosides of the 2′-deoxystreptamine class with high affinity. The regulatory activity, however, strongly depends on the identity of the aminoglycoside. As a prerequisite for the structure determination of riboswitch-ligand complexes we report here the 1H, 15N, 13C and partial 31P chemical shift assignments for the minimal functional 27nt neomycin sensing riboswitch RNA in complex with the 4,5-linked neomycin analog ribostamycin and the 4,6-linked aminoglycoside tobramycin.

Characterization of the targeting signal in mitochondrial β-barrel proteins

Mitochondrial β-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized β-hairpin motif is this long searched for signal. We demonstrate that a synthetic β-hairpin peptide competes with the import of mitochondrial β-barrel proteins and that proteins harbouring a β-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a β-hairpin motif from mitochondrial proteins targets chloroplast β-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the β-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that β-barrel proteins are targeted to mitochondria by a dedicated β-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase.

The First structure of a lantibiotic immunity protein, SpaI from Bacillus subtilis, reveals a novel fold.

Background: The periplasmic lipoprotein SpaI protects the subtilin-producing Bacillus subtilis against its own lantibiotic by an unknown mechanism. Results: The first structure of a lantibiotic immunity protein, SpaI, reveals a novel fold and its membrane-interacting regions. Conclusion: The membrane interaction is important for SpaI-mediated immunity. Significance: The SpaI structure will help to understand the immunity of B. subtilis against subtilin on a structural level. Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.

A Stably Protonated Adenine Nucleotide with a Highly Shifted pKa Value Stabilizes the Tertiary Structure of a GTP-Binding RNA Aptamer

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pKa values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pKa for A nucleotides in single-stranded RNA. This is the largest pKa shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pKa shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pKa shift.

What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin-Sensing Riboswitch.

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.