The structure of the SAM/SAH-binding riboswitch.

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

Adenine protonation enables cyclic-di-GMP binding to cyclic-GAMP sensing riboswitches.

In certain structural or functional contexts, RNA structures can contain protonated nucleotides. However, a direct role for stably protonated nucleotides in ligand binding and ligand recognition has not yet been demonstrated unambiguously. Previous X-ray structures of c-GAMP binding riboswitch aptamer domains in complex with their near-cognate ligand c-di-GMP suggest that an adenine of the riboswitch either forms two hydrogen bonds to a G nucleotide of the ligand in the unusual enol tautomeric form or that the adenine in its N1 protonated form binds the G nucleotide of the ligand in its canonical keto tautomeric state. By using NMR spectroscopy we demonstrate that the c-GAMP riboswitches bind c-di-GMP using a stably protonated adenine in the ligand binding pocket. Thereby, we provide novel insights into the putative biological functions of protonated nucleotides in RNA, which in this case influence the ligand selectivity in a riboswitch.

Inhibitor-Induced Dimerization of an Essential Oxidoreductase from African Trypanosomes.

Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. In vitro and in vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.

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.

Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS.

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.

NMR resonance assignments for the SAM/SAH-binding riboswitch RNA bound to S-adenosylhomocysteine.

Riboswitches are structured RNA elements in the 5'-untranslated regions of bacterial mRNAs that are able to control the transcription or translation of these mRNAs in response to the specific binding of small molecules such as certain metabolites. Riboswitches that bind with high specificity to either S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) are widespread in bacteria. Based on differences in secondary structure and sequence these riboswitches can be grouped into a number of distinct classes. X-ray structures for riboswitch RNAs in complex with SAM or SAH established a structural basis for understanding ligand recognition and discrimination in many of these riboswitch classes. One class of riboswitches-the so-called SAM/SAH riboswitch class-binds SAM and SAH with similar affinity. However, this class of riboswitches is structurally not yet characterized and the structural basis for its unusual bispecificity is not established. In order to understand the ligand recognition mode that enables this riboswitch to bind both SAM and SAH with similar affinities, we are currently determining its structure in complex with SAH using NMR spectroscopy. Here, we present the NMR resonance assignment of the SAM/SAH binding riboswitch (env9b) in complex with SAH as a prerequisite for a solution NMR-based high-resolution structure determination.