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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Molecular Knots as Templates for Protein Engineering:The Story of Lasso PeptidesKok-Phen Yan, Séverine Zirah, Yanyan Li, Christophe Goulard,Rémi Ducasse, Alain Blond, Jean Peduzzi, and Sylvie RebuffatMuséum National d’Histoire Naturelle, Centre National de la Recherche Scientifique,Molécules de Communication et Adaptation des Microorganismes,FRE 3206 CNRS-MNHN, Paris, FranceIntroductionLasso peptides are unusual ribosomally synthesized peptides from bacteria with an originalstructure forming a molecular knot. The N-terminus is covalently linked to the carboxylside chain of an Asp or Glu residue at position 8 or 9, forming a macrolactam ring, which isthreaded by the C-terminal tail, thus forming a lasso. The lasso structure is stabilized bysterical constraints that maintain the C-terminal tail within the ring and/or one or twodisulfide bridges (Figure 1) [1-5].Type 1 Type 2Type 3GAFNCC GSD Y GC A IL GVV CFWIGGTVPFIYPSVEHFGGG AYGNFRATVPQDIFGGSTPRFGPGIPSCWDGNGWLTPCPAWRP 71955 [4] Microcin J25 [1,2] Capistruin [3]BI-32169 [5]Fig. 1. Sequences and topology of type 1 (2 disulfide bridges), 2 (no disulfide bridge) and3 (1 disulfide bridge) lasso peptides. The macrolactam ring, C-terminal tail and bulkyresidues located above and below the ring are indicated in soft grey, grey and dark grey,respectively. The disulfide bridges are shown in black.Microcin J25 (MccJ25, Figure 1) is a lasso peptide produced by E. coli AY25 that exertspotent antimicrobial activity against Salmonella and Escherichia species [6]. The lassostructure of MccJ25 is stabilized by two aromatic residues (F 19 and Y 20 ) located on eachside of the ring. MccJ25 is apportioned into a loop (V 11 -P 16 ) and a ring (Figure 1), whichare involved in the import into target cells via interaction with the siderophore receptorFhuA [7], and in the inhibition of the RNA polymerase, which is its intracellular target [8],respectively. The gene cluster encoding MccJ25 is composed of four genes mcjA, mcjB,mcjC and mcjD. The reconstitution of MccJ25 in vitro from its precursor McjA incubatedwith the maturation enzymes McjB and McjC in the presence of ATP and Mg 2+ [9] pavedthe way for the characterization of the biosynthesis of lasso peptides. We present here ourrecent results on the biosynthesis process of MccJ25 and on structure/activity relationshipsbased on rationally-designed variants of MccJ25.Results and DiscussionSite-directed mutagenesis on the genes encoding the maturation enzymes identified thecatalytic sites involved in the maturation process and the respective roles of McjB andMcjC. McjB is a cysteine protease involved in the cleavage of the leader sequence ofMcjA, while McjC is responsible for both the activation of Glu8 and the subsequentcyclization step. Deletions in the leader region of McjA showed that the 6 last residuesbefore the cleavage site are important for the maturation, in agreement with the results fromLink and coll. [10].34