Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

Abstract

Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo. Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans. SBT3 was inhibited by SBT3PP with a K d of 74 nM for the enzyme-inhibitor complex. With a melting point of 87°C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall. Subtilases (SBTs)2 are found in all kingdoms of life. They constitute the S8 family of serine peptidases (1) and are characterized by a specific arrangement of the aspartate, histidine, and serine residues of the catalytic triad within the active site of the enzyme (2). SBTs include general proteases with relaxed substrate specificity for protein degradation and turnover as well as processing enzymes that cleave selected substrates at highly specific sites (3). Most bacterial SBTs are of the catabolic type including subtilisin E and subtilisin Carlsberg from Bacillus subtilis and Bacillus licheniformis, the prototypical members of the S8A subfamily of subtilases (4). The type-example for subfamily S8B is kexin from Saccharomyces cerevisiae, which was identified as the first SBT from eukaryotes and the first with narrow specificity for dibasic cleavage sites (5). Dibasic cleavage specificity is typically found also in the seven kexin-related proprotein convertases (PCs) in mammals (6). In contrast, all plant SBTs belong to subfamily S8A. They are thus more closely related to bacterial subtilisins than to kexin, but they comprise both, general proteases for protein turnover as well as processing enzymes for limited proteolysis at highly specific sites (7,8).Early structural investigations of subtilisin E showed that it is produced with an N-terminal signal peptide targeting the protein to the periplasmic spac...

Regulation of plant peptide hormones and growth factors by post‐translational modification

“…SBTs remain inactive until the prodomain is cleaved auto-catalytically. For SBT3 from tomato, prodomain cleavage was shown to be controlled by pH, suggesting that the protease remains inactive until the pH drops along the secretory pathway in the trans-Golgi (Meyer et al 2016). The activation of tomato SBT1, on the other hand, requires an additional N-terminal processing event that occurs in the acidic environment of the cell wall (Janzik et al 2000).…”

From structure to function – a family portrait of plant subtilases

“…; Sotokawauchi et al ., ). In contrast to SlSBT3, which shows high specificity for its own prodomain (Meyer et al ., ), cucumisin is also inhibited by distantly related prodomains of SBTs from rice and Arabidopsis (Nakagawa et al ., ). Considering the substrate‐like binding of the prodomain to the active site of the protease, the higher substrate selectivity of SlSBT3 as compared with cucumisin offers an explanation for this difference in specificity.…”

Phytaspase‐mediated precursor processing and maturation of the wound hormone systemin

“…The amino acid residue immediately preceding the TTHT motif (shown to be the N-terminal sequence in mature SBTs and in phytaspases, in particular; Meichtry et al, 1999;Chichkova et al, 2010) is Asp for both new enzymes. As cleavage of the D-TTHT bond for processing of the prodomain occurs in an autocatalytic reaction (Cedzich et al, 2009;Chichkova et al, 2010;Meyer et al, 2016b), the newly identified enzymes are likely to display Asp (D) specificity of hydrolysis. Furthermore, a histidine residue (His331 according to Nt Phytaspase numbering) predicted by structural modeling to bind the P1 Asp of the phytaspase substrate (Vartapetian et al, 2011) is also conserved in both tomato enzymes.…”