Prolyl isomerases in a minimal cell

Abstract

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.

Mycobacterium tuberculosis Cyclophilin A Uses Novel Signal Sequence for Secretion and Mimics Eukaryotic Cyclophilins for Interaction with Host Protein Repertoire

“…However, these pull-down assays and subsequent mass-spectrometric analysis revealed chaperonin GroEL and elongation factor Tu as interacting partners (Table 1). These interactions confirmed earlier reports depicting interaction between prolyl isomerases and chaperones [15]. The possibility of PpiA secretion through the twin-arginine transport system or general secretory pathways was ruled out since PpiA sequence did not possess the characteristic features of the substrates of these pathways [9], [10].…”

“…The cyclophilins belong to an ancient protein family ubiquitous throughout organismal hierarchy [13], [14]. Unlike the eukaryotes, which encode multitude of cyclophilins, most prokaryotes possess a few cyclophilins [15], [16]. The prokaryotic cyclophilins contain a single cyclophilin-like domain (CLD) unlike the large multi-domain eukaryotic counterparts and show a weaker cyclosporin A binding capacity than the eukaryotic cyclophilins [16], [17].…”

Predicting the Minimal Translation Apparatus: Lessons from the Reductive Evolution of Mollicutes

“…In M. genitalium , TIG has two activities: the co-translational folding of nascent polypeptide and a peptidyl-prolyl isomerase activity [47], [48]. Together with DnaK/DnaJ/GrpE and GroEL/GroES, TIG belongs to the essential polypeptide chaperone networking system (see below and [49], [50].…”

Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures

“…1,2 Among organisms with cyclophilins, they are found in every cell and are often present as multiple isoforms (e.g., 17 isoforms exist in humans, eight in Saccharomyces cerevisiae , and two in Escherichia coli ). 3,4 Most, although not all, cyclophilins catalyze the cis – trans isomerization of peptidyl–prolyl bonds.…”