Functional characterization of propeptides in plant subtilases as intramolecular chaperones and inhibitors of the mature protease.
- Publication Type
- Journal contribution (peer reviewed)
- Authors
- Meyer, M., Leptihn, S., Welz, M., Schaller, A.
- Year of publication
- 2016
- Published in
- J. Biol. Chem.
- Band/Volume
- Epub ahead of print/
- DOI
- 10.1074/jbc.M116.744151
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 Kd 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 to 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 auto-inhibitor, 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 auto-inhibited complex relied on a pH-dependent cleavage of the propeptide at Asn38 and Asp54. The remarkable stability of the auto-inhibited 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.