Autoprocessing Mechanism Of An HtrA-like Protease From Thermophilic Brevibacillus Sp.WF146

The serine protease high-temperature requirement A(HtrA)are widely distributed in archaea,bacteria,fungi,plants and animals.HtrAs play a pivotal role in protein quality control in organisms under hostile environments,and contribute to bacterial pathogenicity and remodeling of cell-cell contacts in bacteria by specially degrading damaged proteins.Nowadays,most known HtrAs are from mesophilic bacteria,while little is known about HtrAs from thermophiles that naturally survive at high temperatures.In this study,we firstly obtained the draft genome sequence of Brevibacillus sp.WF146.The strain WF146 genome encodes at least 92 protease genes,including a gene encoding an HtrA-like protease(named HtrAw).The intact form of HtrAw(iHtrAw)consisting of a transmembrane segment(TMS)-containing N-terminal domain,a trypsin-like protease domain,and a C-terminal PDZ domain.Recombinant HtrAw with a fused C-terminal His-tag was produced in E.coli.We found that intact HtrAw could be converted into a short form(sHtrAw)by autocleavage of the peptide bond Ile74-Ser75 downstream of the TMS.HtrAw has the ability to form dimer,trimer,hexamer,and higher order oligomers.The N-terminal domain of HtrAw is not required for enzyme folding,activity,or thermostability,however,the PDZ domain is critical for enzymatic activity of HtrAw.Ca2+,Mg2+,and Mn2+ could enhance the activity and thermostability of HtrAw.Compared with other known HtrAs,HtrAw contains an additional Ca2+-binding Dx[DN]xDG motif.Mutational analysis revealed that the Ca2+-binding Dx[DN]xDG motif contributes to structural stability and enzyme activity of HtrAw,emphasizing the role of Ca2+ in the functional regulation of HtrAw.Secondly,we investigated the processing mechanism of HtrAw.We found that purified iHtrAw itself is unable to cleave the N-terminal domain,but requires denatured or unstructured protein substrates to autoprocess the N-terminal domain in an intermolecular manner,yielding the short form sHtrAw.Mutation at the substrate-binding site in the PDZ domain affects the conversion of iHtrAw to sHtrAw,which suggests that the substrate-binding site in the PDZ domain plays an important role in substrate-induced autoprocessing of the N-terminal domain of HtrAw.Meanwhile,denatured proteins or digested protein substrates could trigger structural change of HtrAw and reduce its thermostability,leading to the autodegradation of the enzyme.Mutation analysis revealed that the autodegradation of the N-termini and PDZ domain was induced by denatured proteins or digested protein substrates binding to PDZ domain.Finally,the physiological role of HtrAw was investigated.In strain WF146,HtrAw exists in both a cell-associated intact form and a cell-free short form;an increase in growth temperature enhanced HtrAw production and the amount of cell-free short form.This suggests that the expression of HtrAw in strain WF146 is temperature-dependent.Meanwhile,an increase in protein substrate concentration in culture medium also promotes HtrAw production and the release of the enzyme into culture medium by autoprocessing the N-terminal domain.When produced in an htrA/htrB double deletion mutant of Bacillus subtilis,iHtrAw localized predominantly to the cell pellet,and the amount of sHtrAw in the culture supernatant increased at elevated temperatures.Moreover,HtrAw increased the heat resistance of the B.subtilis mutant.