The Propeptide is Required for In Vivo Formation of Active Protealysin.

Two structurally distinct N-terminal propeptides are known in thermolysin-like proteases (TLPs). Propeptides of the first type are similar to the prosequence of thermolysin, while the second type propeptides resemble the protealysin propeptide. At the same time, the catalytic domains of all enzymes of the family are highly similar. The available data suggest that the propeptides determine the biological function of TLPs. It was shown that the thermolysin-type propeptides act as folding assistants, can inhibit cognate mature proteins, and influence their secretion. However, the functions of protealysin-like propeptides remain unclear. Here, we studied the effect of the propeptide on protealysin folding for the first time. After heterologous expression in E. coli cells, active enzyme is formed only in the presence of the propeptide either in cis or in trans. Thus, both types of TLP prosequences can function as folding assistants despite their structural dissimilarity and absolutely different interaction with the cognate catalytic domains as indicated by X-ray data.

Crystal structure of the protealysin precursor: insights into propeptide function.

Protealysin (PLN) belongs to the M4 family of peptidases that are commonly known as thermolysin-like proteases (TLPs). All TLPs are synthesized as precursors containing N-terminal propeptides. According to the primary structure of the N-terminal propeptides, the family is divided into two distinct groups. Representatives of the first group including thermolysin and all TLPs with known three-dimensional structures have long prosequences ( approximately 200 amino acids). Enzymes of the second group, whose prototype is protealysin, have short ( approximately 50 amino acids) propeptides. Here, we present the 1.8 A crystal structure of PLN precursor (proPLN), which is the first three-dimensional structure of a TLP precursor. Whereas the structure of the catalytic domain of proPLN is similar overall to previously reported structures of mature TLPs, it has specific features, including the absence of calcium-binding sites, and different structures of the N-terminal region and substrate-binding site. PLN propeptide forms a separate domain in the precursor and likely acts as an inhibitor that blocks the substrate-binding site and fixes the "open" conformation of the active site, which is unfavorable for catalysis. Furthermore the conserved PPL motif identified in our previous studies directly interacts with the S' subsites of the active center being a critical element of the propeptide-catalytic domain interface. Comparison of the primary structures of TLPs with short propeptides suggests that the specific features revealed in the proPLN crystal structure are typical for all protealysin-like enzymes. Thus, such proteins can be considered as a separate subfamily of TLPs.

Processing of protealysin precursor.

Protealysin, a protease previously described by us in Serratia proteamaculans, belongs to the group of thermolysin-like proteases (TLPs) that differ from classical TLPs by the precursor structural organization. The propeptide of protealysin precursor has no significant structural similarity to the propeptides of most TLPs. The functions of protealysin-like precursors and mechanisms of their action remain unclear. We studied the pathway of protealysin precursor processing in vitro using standard approaches: modification of the catalytic site and monitoring immobilized precursor maturation. The Glu(113) --> Ala substitution inhibited the precursor maturation, which pointed to the autocatalytic processing. The mutant precursor exposure to active protealysin converted it to the mature enzyme, thus, indicating the intermolecular processing. Intermolecular processing of the mutant protein by other proteases such as thermolysin or subtilisin is also possible. The intact protealysin precursor was efficiently autoprocessed in solution but not after immobilization. These data indicate that the processing of protealysin precursor differs from that of classical TLPs. The protealysin propeptide is cleaved by an autocatalytic or heterocatalytic intermolecular mechanism and is most likely not removed intramolecularly.