An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Abstract

The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cisltrans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatieally active fragment could be isolated after proteolysis by subtifisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni 2+-NTA column. Subsequent digestion with subtifisin and anionexchange chromatography yielded a TF fragment encompassing amino acids Gin-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 ~1-1 s -l could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrofide FK506.

The Escherichia coli trigger factor

“…Proteolytic fragments of 12.8 kDa (Va1132 to Glu247) and 11.8 kDa (Arga45 to Glu 251), respectively, displayed a specific PPIase activity similar to that of full-length trigger factor. An even smaller recombinant fragment comprising only 102 amino acids (Glu148 to Thr249) also displayed full PPIase activity [18]. The size of this PPIase domain is in good agreement with the size of the prototype FKBP12.…”

“…These homology predictions were recently confirmed by functional analyses of trigger factor fragments obtained by limited proteolysis with endoproteinase Glu-C (V8) [17] and subtilisin [18]. Proteolytic fragments of 12.8 kDa (Va1132 to Glu247) and 11.8 kDa (Arga45 to Glu 251), respectively, displayed a specific PPIase activity similar to that of full-length trigger factor.…”

“…While a proteolytic fragment of 14 kDa apparent molecular weight starting with the authentic N-terminus of trigger factor has been reported [18], the polypeptide chain C-terminal to the PPIase domain is highly susceptible to proteolysis [17,18]. The functional roles of these domains remain to be elucidated.…”

Structure-Function Analysis of PrsA Reveals Roles for the Parvulin-like and Flanking N- and C-terminal Domains in Protein Folding and Secretion in Bacillus subtilis

“…However, the catalytic activity of PrsA was fairly weak. Much higher activities have been reported for the E. coli trigger factor, human Pin1 and E. coli PpiD (4,13,42), whereas the activity of E. coli SurA is comparable to that of PrsA (8). Because PrsA-PPI protein containing only the parvulin-like region catalyzed the proline-limited folding in a similar manner as the full-length PrsA, the parvulin-like region is clearly responsible for the activity.…”

Trigger Factor-Mediated Prolyl Isomerization Influences Maturation of the Streptococcus pyogenes Cysteine Protease

“…In addition, the N-terminal 118 amino acids are required for binding to ribosomes (17). The central domain has homology to the family of FK506-binding PPIase proteins (13,16,35). This class of proteins can catalyze the isomerization of the peptide bond preceding a prolyl res-idue, and the central domain of trigger factor can function independently in vitro as an active PPIase (16,35).…”