Christoph Wiedemann

Stereochemical assignments of thiazoline-containing cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides, are often complicated due to the propensity of the exomethine and C-4 chiral centers of thiazolines to epimerize under basic and acidic conditions. In this work, we re-evaluate the proposed configuration of the leucine-thiazoline moiety of the cyanobactin-like cyclopeptide keenamide A. Using a fast and adaptable strategy for the synthesis of thiazoline-containing cyclopeptides, we synthesized four possible keenamide A stereoisomers, one of which was oxidized to mollamide C, the thiazole analogue of keenamide A. Comparison of the NMR spectra of synthetic keenamide A stereoisomers with that of natural keenamide A combined with X-ray crystallographic analysis indicates that the originally proposed (R)-configuration of the thiazoline ring of keenamide A must be revised to (S)-configuration. This work highlights the power of synthetic methods to inform the structure elucidation and structural revision of natural products, especially in cases where isolation and purification of natural material are not readily achieved.

Bacterially produced antimicrobial peptides (AMPs), or bacteriocins, play key roles in shaping microbial communities via interspecies competition. Unlike the more temporally dynamic gut microbiome, the oral microbiome exhibits long-term stability and is preserved into deep time in dental calculus, enabling evolutionary analysis across time. Here, we combine metagenomics, structural modeling, and experimental validation to investigate AMP diversity in ancient and modern dental biofilms from humans, Neanderthals, and nonhuman primates spanning 100,000 years. Using our newly developed platform, AMPcombi, we uncover evolutionary trajectories of bacteriocins and elucidate their ecological functions. Among these, we identify a conserved family of Actinomyces-derived defensin-like peptides, termed actifensins, present across all time periods. Phylogenetic, structural, and functional analyses revealed shared ancestry and adaptive diversification between ancient (paleo-) and modern actifensins, with evidence of positive selection and maintained antimicrobial activity. Our findings position the oral microbiome as a valuable reservoir for natural product discovery. In the face of rising antimicrobial resistance, evolutionary insights into AMP function open a door to next-generation therapeutics. AMPcombi streamlines this process, linking ancient biomolecules with biotechnology.

Macrophage infectivity potentiator (MIP) proteins, found in pro- and eukaryotic pathogens, influence microbial virulence, host cell infection, pathogen replication, and dissemination. MIPs share an FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain, making them attractive targets for inhibitor development. We determined high-resolution crystal structures of Burkholderia pseudomallei and Trypanosoma cruzi MIPs in complex with fluorinated pipecolic acid inhibitors. The inhibitor binding profiles in solution were compared across B. pseudomallei, T. cruzi, and Legionella pneumophila MIPs using ¹H, ¹⁵N, and ¹⁹F NMR spectroscopy. Demonstrating the versatility of fluorinated ligands for characterizing inhibitor complexes, ¹⁹F NMR spectroscopy identified differences in ligand binding dynamics across MIPs. EPR spectroscopy and SAXS further revealed inhibitor-induced global structural changes in homodimeric L. pneumophila MIP. This study demonstrates the importance of integrating diverse methods to probe protein dynamics and provides a foundation for optimizing MIP-targeted inhibitors in this structurally conserved yet dynamically variable protein family.

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro. In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to study this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

Macrophage infectivity potentiator (MIP) proteins are present in many microbial pathogens, both of pro- and of eukaryotic origin. MIP proteins play important roles in microbial virulence, facilitating processes like host cell infection, pathogen replication and dissemination. While their domain architecture differs in MIPs, they all share a FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain. Due to its high conservation, the FKBP domain has been a target for inhibitor development, including pipecolic acid derivatives. Here, we determined the high-resolution crystal structures of Burkholderia pseudomallei and Trypanosoma cruzi MIP in complex with fluorinated pipecolic acid inhibitors. Furthermore, we compared the inhibitor binding profiles of MIP proteins from B. pseudomallei, T. cruzi and Legionella pneumophila MIP using 1H, 15N-NMR spectroscopy. Additionally, 19F NMR was used to gauge inhibitor binding to the different MIP proteins. Complementary approaches including EPR spectroscopy and SAXS were used to investigate the global structural changes of homodimeric L. pneumophila MIP upon inhibitor binding. Our data provide a foundation for future inhibitor optimization and advance our understanding of the unexpected dynamic variability within a structurally conserved protein family.

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP₂ binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP₂-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

Herein we report a mild, efficient, and epimerization‐free method for the synthesis of peptide‐derived 2‐thiazolines and 5,6‐dihydro‐4H‐1,3‐thiazines based on a cyclodesulfhydration ofN‐thioacyl‐2‐mercaptoethylamine orN‐thioacyl‐3‐mercaptopropylamine derivatives. The described reaction can be easily carried out in aqueous solutions at room temperature and it is triggered by change of the pH, leading to complex thiazoline or dihydrothiazine derivatives without epimerization in excellent to quantitative yields. The new method was applied in the total synthesis of the marine metabolite mollamide F, resulting in the revision of its stereochemistry.

In diesem Bericht beschreiben wir eine milde, effiziente und epimerisierungsfreie Methode für die Synthese 2‐thiazolin‐ und 5,6‐dihydro‐4H‐1,3‐thiazinhaltiger Peptide durch Cyclodesulfhydrierung entsprechenderN‐thioacylierter 2‐Mercaptoethylamin‐ oder 3‐Mercaptopropylaminderivate. Die beschriebene Reaktion wird durch eine Änderung des pH‐Wertes ausgelöst, kann in wässrigen Lösungen und bei Raumtemperatur durchgeführt werden und führt in hervorragenden bis quantitativen Ausbeuten zu komplexen Thiazolin‐ und Dihydrothiazinderivaten. Die neue Methode kam erfolgreich bei der Totalsynthese des marinen Naturstoffes Mollamid F zum Einsatz, was eine Revision seiner Stereochemie zur Folge hatte.

Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila , the causative agent of Legionnaires’ disease and protozoans such as Trypanosoma cruzi . All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl- cis/trans - isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella pneumophila protein ( Lp MIP) have additional appendage domains of mostly unknown function. In full- length, homodimeric Lp MIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of Lp MIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of Lp MIP and Tc MIP at 1.49 and 1.34 Å, respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms.