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 ((N)DDs) as well as of an (DD)-D-N-(DD)-D-C 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 riboswitchesthe so-called SAM/SAH riboswitch classbinds 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

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 reactionsand 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