Substrate interactions guide cyclase engineering and lasso peptide diversification.

Autor: Barrett SE; Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA.; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA., Yin S; Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA., Jordan P; Lassogen, Inc., San Diego, CA, USA., Brunson JK; Lassogen, Inc., San Diego, CA, USA., Gordon-Nunez J; Lassogen, Inc., San Diego, CA, USA., Costa Machado da Cruz G; Lassogen, Inc., San Diego, CA, USA., Rosario C; Lassogen, Inc., San Diego, CA, USA., Okada BK; Lassogen, Inc., San Diego, CA, USA., Anderson K; Lassogen, Inc., San Diego, CA, USA., Pires TA; Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA.; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA., Wang R; Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA.; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA., Shukla D; Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA.; Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA.; Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA.; Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL, USA., Burk MJ; Lassogen, Inc., San Diego, CA, USA. mburk@lassogen.com., Mitchell DA; Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA. douglasm@illinois.edu.; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA. douglasm@illinois.edu.; Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, IL, USA. douglasm@illinois.edu.
Jazyk: angličtina
Zdroj: Nature chemical biology [Nat Chem Biol] 2024 Sep 11. Date of Electronic Publication: 2024 Sep 11.
DOI: 10.1038/s41589-024-01727-w
Abstrakt: Lasso peptides are a diverse class of naturally occurring, highly stable molecules kinetically trapped in a distinctive [1]rotaxane conformation. How the ATP-dependent lasso cyclase constrains a relatively unstructured substrate peptide into a low entropy product has remained a mystery owing to poor enzyme stability and activity in vitro. In this study, we combined substrate tolerance data with structural predictions, bioinformatic analysis, molecular dynamics simulations and mutational scanning to construct a model for the three-dimensional orientation of the substrate peptide in the lasso cyclase active site. Predicted peptide cyclase molecular contacts were validated by rationally engineering multiple, phylogenetically diverse lasso cyclases to accept substrates rejected by the wild-type enzymes. Finally, we demonstrate the utility of lasso cyclase engineering by robustly producing previously inaccessible variants that tightly bind to integrin αvβ8, which is a primary activator of transforming growth factor β and, thus, an important anti-cancer target.
Competing Interests: Competing interests: M.J.B. and D.A.M. are co-founders of and own stock in Lassogen, Inc. S.E.B. and D.A.M. are inventors on a provisional patent application filed by the University of Illinois at Urbana-Champaign covering lasso cyclase engineering (US Provisional Application Ser. No. 63/673,853). M.J.B., P.A.J. and G.C.M.d.C. are inventors on a provisional patent application filed by Lassogen, Inc. for the design of lasso peptide integrin binders (US Provisional Application No. 63/612,957). The other authors declare no competing interests.
(© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE
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