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

The boundaries of the structural domains in peptide synthetases and the con formational changes related to catalysis were investigated by limited prote olysis of tyrocidine synthetase 1 (TY1). Four regions sensitive to proteoly sis were detected (cleavage site at Arg13, Arg424, Arg509 and Arg602) that, in addition to an N-terminal extension, accurately delineate the domain bo undaries of the adenylate-forming domain, the aminoacyl carrier domain, and the epimerisation domain limited proteolysis of an active N-terminal trunc ated deletion mutant, His(6)Delta TY1, generated two stable and structurall y 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 proteolysis . 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 t he X-ray structure of the homologous firefly luciferase. Sequence analysis has shown that the respective linker contains conserved residues, whereas t he linker regions connecting the structural domains are of low homology wit h a significant content of Pro, Ala, Glu and polar residues. A combination of kinetic and proteolytic studies using ATP analogues with substitutions i n 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 t he beta,gamma-pyrophosphate moiety of ATP to adopt the proper active-site c onformation. These data substantiate the observation that peptide synthetas es undergo a series of conformational changes in the process of adenylate f ormation and product release. (C) 1999 Academic Press.