Conformations of Macrocyclic Peptides Sampled by Nuclear Magnetic Resonance: Models for Cell-Permeability.

The biological activities and pharmacological properties of peptides and peptide mimetics are determined by their conformational states. Therefore, a detailed understanding of the conformational landscape is crucial for rational drug design. Nuclear magnetic resonance (NMR) is the only method for structure determination in solution. However, it remains challenging to determine the structures of peptides using NMR because of very weak nuclear Overhauser effects (NOEs), the semiquantitative nature of the rotating frame Overhauser effect (ROE), and the low number of NOEs/ROEs in N-methylated peptides. In this study, we introduce a new approach to investigating the structures of modified macrocyclic peptides. We utilize exact NOEs (eNOEs) in viscous solvent mixtures to replicate various cellular environments. eNOEs provide detailed structural information for highly dynamic modified peptides. Structures of high precision were obtained for cyclosporin A, with a backbone atom rmsd of 0.10 Å. Distinct conformational states in different environments were identified for omphalotin A (OmphA), a fungal nematotoxic and multiple backbone N-methylated macrocyclic peptides. A model for cell-permeation is presented for OmphA, based on its structures in polar, apolar, and mixed polarity solvents. During the transition from a polar to an apolar environment, OmphA undergoes a rearrangement of its H-bonding network, accompanied by a cis to trans isomerization of the ω torsion angle within a type VIa ß-turn. We hypothesize that the kinetics of these conformational transitions play a crucial role in determining the membrane-permeation capabilities of OmphA.

Genome sequences of Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana and the identification of biosynthetic gene clusters for fungal peptide natural products.

In recent years, a variety of fungal cyclic peptides with interesting bioactivities have been discovered. For many of these peptides, the biosynthetic pathways are unknown and their elucidation often holds surprises. The cyclic and backbone N-methylated omphalotins from Omphalotus olearius were recently shown to constitute a novel class (borosins) of ribosomally synthesized and posttranslationally modified peptides, members of which are produced by many fungi, including species of the genus Rhizopogon. Other recently discovered fungal peptide macrocycles include the mariannamides from Mariannaea elegans and the backbone N-methylated verrucamides and broomeanamides from Myrothecium verrucaria and Sphaerostilbella broomeana, respectively. Here, we present draft genome sequences of four fungal species Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana. We screened these genomes for precursor proteins or gene clusters involved in the mariannamide, verrucamide, and broomeanamide biosynthesis including a general screen for borosin-producing precursor proteins. While our genomic screen for potential ribosomally synthesized and posttranslationally modified peptide precursor proteins of mariannamides, verrucamides, broomeanamides, and borosins remained unsuccessful, antiSMASH predicted nonribosomal peptide synthase gene clusters that may be responsible for the biosynthesis of mariannamides, verrucamides, and broomeanamides. In M. verrucaria, our antiSMASH search led to a putative NRPS gene cluster with a predicted peptide product of 20 amino acids, including multiple nonproteinogenic isovalines. This cluster likely encodes a member of the peptaibols, an antimicrobial class of peptides previously isolated primarily from the Genus Trichoderma. The nonribosomal peptide synthase gene clusters discovered in our screenings are promising candidates for future research.

Structural and Functional Analysis of Peptides Derived from KEX2-Processed Repeat Proteins in Agaricomycetes Using Reverse Genetics and Peptidomics.

Bioactivities of fungal peptides are of interest for basic research and therapeutic drug development. Some of these peptides are derived from "KEX2-processed repeat proteins" (KEPs), a recently defined class of precursor proteins that contain multiple peptide cores flanked by KEX2 protease cleavage sites. Genome mining has revealed that KEPs are widespread in the fungal kingdom. Their functions are largely unknown. Here, we present the first in-depth structural and functional analysis of KEPs in a basidiomycete. We bioinformatically identified KEP-encoding genes in the genome of the model agaricomycete Coprinopsis cinerea and established a detection protocol for the derived peptides by overexpressing the C. cinerea KEPs in the yeast Pichia pastoris. Using this protocol, which includes peptide extraction and mass spectrometry with data analysis using the search engine Mascot, we confirmed the presence of several KEP-derived peptides in C. cinerea, as well as in the edible mushrooms Lentinula edodes, Pleurotus ostreatus, and Pleurotus eryngii. While CRISPR-mediated knockout of C. cinerea kep genes did not result in any detectable phenotype, knockout of kex genes caused defects in mycelial growth and fruiting body formation. These results suggest that KEP-derived peptides may play a role in the interaction of C. cinerea with the biotic environment and that the KEP-processing KEX proteases target a variety of substrates in agaricomycetes, including some important for mycelial growth and differentiation. IMPORTANCE Two recent bioinformatics studies have demonstrated that KEX2-processed repeat proteins are widespread in the fungal kingdom. However, despite the prevalence of KEPs in fungal genomes, only few KEP-derived peptides have been detected and studied so far. Here, we present a protocol for the extraction and structural characterization of KEP-derived peptides from fungal culture supernatants and tissues. The protocol was successfully used to detect several linear and minimally modified KEP-derived peptides in the agaricomycetes C. cinerea, L. edodes, P. ostreatus, and P. eryngii. Our study establishes a new protocol for the targeted search of KEP-derived peptides in fungi, which will hopefully lead to the discovery of more of these interesting fungal peptides and allow a further characterization of KEPs.