Molecular Basis Of Processing And Maturation Of Thermophilic WF146Protease

Subtilisin-like serine proteases (subtilases) are widely distributed in bacteria, archaea and eukaryotes, and play important roles in a variety of biological processes such as protein catabolism and zymogen activation. Extracellular subtilases are generally produced as inactive precursors, which should undergo maturation to release enzyme activity. The stepwise maturation process enables precise regulation of the activity spatially and temporally. Thermophilic subtilases have attracted considerable attention because of their contributions to the understanding of temperature adaptation mechanism of enzymes, as well as their industrial potential. Investigations on the maturation process of subtilases adapted to different temperatures not only help us to further understand their temperature adaptation strategies but also provide valuable information for preparation of active subtilases of industrial potential.WF146protease is a thermophilic subtilase secreted by thermophile Bacillus sp. WF146, and its precursor contains a signal peptide, an N-terminal propeptide and a mature domain. By using E. coli BL21as the host, the recombinant proform of WF146protease (43kDa) was expressed as soluble form, which could effectively convert to active mature form (32kDa) by processing its N-terminal propeptide when incubated at high temperatures (e.g.,60℃). The purpose of this study is to elucidate the molecular basis of processing and maturation of WF146protease.We first investigated possible maturation pathway(s) of WF146protease. Active site mutants of this enzyme were constructed to probe possible processed products/intermediates produced during the maturation process. In contrast to wild type WF146protease, the active site mutant S249A was unable to mature, suggesting that the active site is required for the autoprocessing of the N-terminal propeptide. In the case of another active site mutant S249C, it could autoprocess to a33-kDa intermediate, but was unable to further convert to the32-kDa mature form. When wild type proform was incubated at60℃with the inhibitor PMSF, the33-kDa intermediate accumulated during the maturation process. Our results suggest that the proform of WF146protease autocatalytically converts to the mature form via an intermediate, and the autoprocessing of the N-terminal propeptide occurs in a stepwise manner. First, the main part of the N-terminal propeptide (N*) is cis-processed by the active site of the mature domain of the proform, generating the intermediate. Subsequently, the linker peptide, which locates between N*and the mature domain in the proform, is truncated from the intermediate, generating the mature enzyme. In addition, the auto-catalytic maturation of WF146protease could also proceed via another pathway, which is initiated by trans-processing of the linker peptide between N*and the mature domain. Moreover, unlike most reported subtilases, the maturation of WF146protease occurs not only auto-catalytically but also hetero-catalytically. By using circular dichroism spectra analysis, we found that WF146protease undergoes subtle structural adjustments during the maturation process. Limited proteolysis revealed that, in comparison with the mature domain, the N-terminal propeptide of the proform is more susceptible to proteolytic degradation, allowing the proform to convert to mature form by trans-processing of the propeptide.Secondly, the role of metal ions (e.g., Ca2+) in the maturation of WF146protease was investigated. Amino acid sequence alignment of WF146protease and its closely related homologs (～68%identity) with known crystal structures, such as subtilisin S41and sphericase, revealed that WF146protease contains at least four Ca2+-binding sites. Besides contributing to the thermostability and activity of WF146protease, Ca2+-binding is important for maturation of the enzyme. When incubated at60℃under chelating conditions (e.g.,2mM EDTA), the proform of WF146protease could not convert into mature form, but was cis-processed into a unique propeptide:intermediate complex capable of re-synthesis of the proform. These results indicate that Ca2+-binding is required for routing the proform of WF146protease to the maturation pathway.Finally, we investigated the roles of the N-terminal propeptide and the linker peptide in enzyme folding, maturation and activity. Our results suggest that the N-terminal propeptide functions both as an intramolecular chaperone to assist the folding of the mature domain, and as a potent inhibitor of the mature WF146protease. We noticed that there is a cluster of five Glu residues in the12-amino acid residue linker peptide. Simultaneous substitution of the five acidic Glu residues by other types of amino acid residues (e.g., Gln, Ala, Gly or Lys) did not influence enzyme activity, but accelerated the maturation process of the enzyme. We postulate that this acidic Glu cluster contributes to the structural adjustment of the proform during the maturation process. Although the linker peptide has no significant influence on the enzyme folding and enzyme activity, its acidic Glu cluster may play an important role in regulating the maturation of the enzyme, allowing the enzyme to release its activity at proper time and space.