Multivalent Interactions between an Aromatic Helical Foldamer and a DNA G‐Quadruplex in the Solid State

Quinoline‐based oligoamide foldamers have been identified as a potent class of ligands for G‐quadruplex DNA. Their helical structure is thought to target G‐quadruplex loops or grooves and not G‐tetrads. We report a co‐crystal structure of the antiparallel hairpin dimeric DNA G‐quadruplex (G₄T₄G₄)₂ with tetramer 1 —a helically folded oligo‐quinolinecarboxamide bearing cationic side chains—that is consistent with this hypothesis. Multivalent foldamer–DNA interactions that modify the packing of (G₄T₄G₄)₂ in the solid state are observed.     

An Iterative O‐Methyltransferase Catalyzes 1,11‐Dimethylation of Aspergillus fumigatus Fumaric Acid Amides

S‐adenosyl‐l ‐methionine (SAM)‐dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11‐dimethyl esters of fumaryl‐l ‐tyrosine and fumaryl‐l ‐phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size‐exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.     

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post‐translational modification of the precursor McjA by the ATP‐dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three‐dimensional folding of MccJ25. We further showed that the N‐terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43–S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23–Q34 in the N‐terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.     

Tracking Protein S‐Fatty Acylation with Proteomics

Recently, Hang and co‐workers developed “acyl‐PEG exchange” (APE), which allows the investigation of protein S‐fatty acylation with mass‐tag labelling and gel electrophoresis, methods that are accessible to any biochemistry laboratory.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post‐translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C‐terminal “follower” sequence, rather than the canonical N‐terminal “leader” sequence. We report herein the structural and biochemical characterization of BotP, a leucyl‐aminopeptidase‐like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N‐terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn²⁺ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro‐ mycin.     

DNA Antenna Tile‐Associated Deoxyribozyme Sensor with Improved Sensitivity

Some natural enzymes increase the rate of diffusion‐limited reactions by facilitating substrate flow to their active sites. Inspired by this natural phenomenon, we developed a strategy for efficient substrate delivery to a deoxyribozyme (DZ) catalytic sensor. This resulted in a three‐ to fourfold increase in sensitivity and up to a ninefold improvement in the detection limit. The reported strategy can be used to enhance catalytic efficiency of diffusion‐limited enzymes and to improve sensitivity of enzyme‐based biosensors.     

DNA‐Templated Synthesis of Perylenediimide Stacks Utilizing Abasic Sites as Binding Pockets and Reactive Sites

DNA is considered to be a promising biomolecule as a template and scaffold for arranging and organizing functional molecules on the nanoscale. The construction and evaluation of DNAs containing multiple functional molecules that are useful for optoelectronic devices and sensors has been studied. In this paper we report the efficient incorporation of perylenediimide (PDI) units into DNA by using abasic sites both as binding sites and as reactive sites and the construction of PDI stacks within the DNA structure, accomplished through the preorganization of the PDI units in the hydrophobic pocket within the DNA. Our approach could become a valuable method for construction of DNA/chromophore hybrid structures potentially useful for the design of DNA‐based devices and biosensors.