engleski

Probing the Domain Structure and Ligand-Induced Conformational Changes by Limited Proteolysis of Tyrocidine Synthetase 1

Limited proteolysis has been applied to define boundaries of stable domains of peptide synthetases and to identify linker regions. Tyrocidine synthetase 1 was used as a model system and exposed to trypsin, proteinase K and subtilisin. Four cleavage regions were detected, which define an N-terminal extension, the adenylate forming domain, the aminoacyl carrier domain, and the epimerization domain. A substrate protected site was located within the adenylate domain corresponding to a peptide stretch only partially visible in the X-ray analysis of the homologous firefly luciferase. While this linker region contained conserved residues, the other linker regions are of low homology with a significant content of Pro, Ala and Glu and polar residues. A segment of the N-terminal 13 (trypsin) or 23 (proteinase K) amino acids was rapidly released. A respective 17 amino acid deletion fragment of the adenylation domain was constructed as His6-fusion and found to be fully active in activation of the substrate Phe. Likewise, the aminoacyl carrier domain was expressed as a 72-peptide fused to maltose binding protein, and could be released as a stable fragment by cleavage with factor Xa. Tryptic cleavage sites of multidomain peptide synthetases including ACV synthetase, enniatin synthetase, and cyclosporin synthetase revealed identical susceptible regions, and additional sites adjacent to N-methyl-transferase domains. These data accurately define domain boundaries in peptide synthetases and provide evidence that the epimerization domain and the homologous condensation domain are autonomous regions stable against limited proteolysis.

Peptide synthetases; limited proteolysis; domain organization; linker

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