| Autor: |
Naudin EA; Institut de Science et d'Ingénierie Supramoléculaires (ISIS), International Center for Frontier Research in Chemistry (icFRC), University of Strasbourg, CNRS (UMR 7006), Strasbourg 67000, France., McEwen AG; Integrated Structural Biology Platform, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), INSERM (U1258), University of Strasbourg, Illkirch 67404, France., Tan SK; Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94158-9001, United States., Poussin-Courmontagne P; Integrated Structural Biology Platform, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), INSERM (U1258), University of Strasbourg, Illkirch 67404, France., Schmitt JL; Institut de Science et d'Ingénierie Supramoléculaires (ISIS), International Center for Frontier Research in Chemistry (icFRC), University of Strasbourg, CNRS (UMR 7006), Strasbourg 67000, France., Birck C; Integrated Structural Biology Platform, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), INSERM (U1258), University of Strasbourg, Illkirch 67404, France., DeGrado WF; Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94158-9001, United States., Torbeev V; Institut de Science et d'Ingénierie Supramoléculaires (ISIS), International Center for Frontier Research in Chemistry (icFRC), University of Strasbourg, CNRS (UMR 7006), Strasbourg 67000, France. |
| Abstrakt: |
The design of catalytic proteins with functional sites capable of specific chemistry is gaining momentum and a number of artificial enzymes have recently been reported, including hydrolases, oxidoreductases, retro-aldolases, and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation that possesses no stringent restrictions to the amino acid composition at the ligation junction. We report here the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied a minimalist approach to rationally design an oxyanion hole within a small cavity that contains an adjacent thiol nucleophile. The N-terminus of the α-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition-elimination reactions at the acyl group. Cysteine acting as a principal catalytic residue was introduced at the second residue position of the α-helix N-terminus in a designed three-α-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide- α thioesters as acyl-donors, we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation, and peptide ligation. |
