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J Mol Biol. 1999 Apr 23;288(1):129-40.

Probing the domain structure and ligand-induced conformational changes by limited proteolysis of tyrocidine synthetase 1.

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Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Berlin, Germany.


The boundaries of the structural domains in peptide synthetases and the conformational changes related to catalysis were investigated by limited proteolysis of tyrocidine synthetase 1 (TY1). Four regions sensitive to proteolysis were detected (cleavage site at Arg13, Arg424, Arg509 and Arg602) that, in addition to an N-terminal extension, accurately delineate the domain boundaries of the adenylate-forming domain, the aminoacyl carrier domain, and the epimerisation domain. Limited proteolysis of an active N-terminal truncated deletion mutant, His6DeltaTY1, generated two stable and structurally independent subunits, corresponding to the subdomains of the adenylation domain. The structural integrity of the carrier domain was substantiated by its resistance to proteolytic degradation. Evidence is provided that the C-terminal "spacer" region with epimerising and/or condensing activity folds into an autonomous domain stable against degradation by limited proteoly sis. In the presence of substrates, reduced susceptibility to proteolysis was observed in the linker region connecting the subdomains of the adenylation domain, and corresponding to a peptide stretch of low electron density in the X-ray structure of the homologous firefly luciferase. Sequence analysis has shown that the respective linker contains conserved residues, whereas the linker regions connecting the structural domains are of low homology with a significant content of Pro, Ala, Glu and polar residues. A combination of kinetic and proteolytic studies using ATP analogues with substitutions in the phosphate chain, AMP-PcP, AMP-PNP and AMP-cPP, strongly suggests that the generation of a productive complex is associated with the ability of the beta, gamma-pyrophosphate moiety of ATP to adopt the proper active-site conformation. These data substantiate the observation that peptide synthetases undergo a series of conformational changes in the process of adenylate formation and product release.

[Indexed for MEDLINE]

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