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Regulation of an Intracellular Subtilisin Protease Activity by a Short Propeptide Sequence Through an Original Combined Dual Mechanism

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Regulation of an Intracellular Subtilisin Protease Activity by a Short Propeptide Sequence Through an Original Combined Dual Mechanism Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism Michael Gamblea,1, Georg Künzea,1,2, Eleanor J. Dodsonb, Keith S. Wilsonb,3, and D. Dafydd Jonesa,3 aSchool of Biosciences, Cardiff University, Cardiff CF10 3AT, United Kingdom; and bStructural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom Edited by Robert Huber, Max Planck Institute for Biochemistry, Planegg-Martinsried, Germany, and approved January 4, 2011 (received for review September 23, 2010) A distinct class of the biologically important subtilisin family of proteinase activity (18, 19). However, little is known regarding serine proteases functions exclusively within the cell and forms the crucial feature of how their activity is regulated posttransla- a major component of the bacilli degradome. However, the mode tionally within the cell, where control of protease activity is vital and mechanism of posttranslational regulation of intracellular to prevent the untimely breakdown of crucial cellular protein protease activity are unknown. Here we describe the role played components. This is exemplified by the harmful effects of intra- by a short N-terminal extension prosequence novel amongst the cellular expression of bacilli ESPs to the host cell (20). subtilisins that regulates intracellular subtilisin protease (ISP) activ- The ISPs are close relatives of the bacilli ESPs, with 40–50% ity through two distinct modes: active site blocking and catalytic sequence identity (21). Despite this, their sequences have a num- triad rearrangement. The full-length proenzyme (proISP) is inactive ber of distinctive features (Fig. 1 A and B) that translate into until specific proteolytic processing removes the first 18 amino tertiary and quaternary structural features unique amongst the acids that comprise the N-terminal extension, with processing subtilisins, including being dimeric (Fig. 1C) (21). Whereas a appearing to be performed by ISP itself. A synthetic peptide corre- dimeric structure has also been observed for the multidomain sponding to the N-terminal extension behaves as a mixed non- tomato subtilase (22), dimerization occurs via a separate PA K μ competitive inhibitor of active ISP with a i of 1 M. The structure domain to the catalytic domain. In contrast, the catalytic domain of the processed form has been determined at 2.6 Å resolution together with a short C-terminal arm mediate dimerization in and compared with that of the full-length protein, in which the ISP (21). A key feature of the ISPs is the replacement of the N-terminal extension binds back over the active site. Unique to classical “foldase” prodomain with a short (16–25 residue) ISP, a conserved proline introduces a backbone kink that shifts N-terminal extension (Fig. 1A), which has no sequence homology the scissile bond beyond reach of the catalytic serine and in addi- B tion the catalytic triad is disrupted. In the processed form, access with the ESP prodomains (Fig. 1 ) and is not involved in the to the active site is unblocked by removal of the N-terminal exten- folding process (20). The LIPY/F sequence motif in the extension B sion and the catalytic triad rearranges to a functional confor- is strictly conserved (Fig. 1 ), hinting at a common role. Posttran- mation. These studies provide a new molecular insight concerning slational removal of the proISP extension has been reported the mechanisms by which subtilisins and protease activity as a in a number of organisms (13, 15, 16, 23) but the implications whole, especially within the confines of a cell, can be regulated. of this event remained unknown until the structure of the full- length ISP from Bacillus clausii was determined in our labora- posttranslational modification ∣ protease regulation ∣ tories (21). The structure suggests that the extension acts as proprotein activation ∣ protease structure an inbuilt inhibitor of activity by binding back over and so block- ing the active site. The LIPY/F motif plays a key role with the ubtilisins are serine endopeptidases ubiquitous in nature, proline introducing a bulge that shifts the scissile peptide Sspread across the eubacteria, archaebacteria, eukaryotes, bond beyond the reach of the catalytic serine, preventing direct and viruses (1). They play a variety of important biological roles binding to the active site (Fig. 1D). that include specific posttranslational processing of hormones Here we describe the role of precise proteolytic processing (2, 3), virulence and infection factors in various pathogens (4, 5), of the N-terminal extension in regulating the activity of the and as nonspecific digestive proteases (1). The distinctive primary B. clausii ISP. Comparison of the recently determined struc- structure features common to the majority of subtilisins, and ture of the full-length protein (proISPS250A) with the processed typified by the bacilli extracellular subtilisins (ESP), comprise a form (NΔ18-ISPS250A) determined here reveals the original, dual N-terminal signal sequence with an adjacent prodomain required approach by which this extension inhibits protease activity. for correct folding of the mature, catalytic domain (6–8) (Fig. 1A). Both the signal sequence and the prodomain are posttranslation- ally removed, the latter autocatalytically. The bacilli ESPs have Author contributions: M.G., K.S.W., and D.D.J. designed research; M.G., G.K., and E.J.D. performed research; M.G., G.K., E.J.D., K.S.W., and D.D.J. analyzed data; and M.G., a broad substrate specificity that suites their role as scavenging G.K., E.J.D., K.S.W., and D.D.J. wrote the paper. proteases, and their robustness to harsh environments coupled The authors declare no conflict of interest. with extensive protein engineering has resulted in their exploita- This article is a PNAS Direct Submission. tion by industry for a variety of applications, in particular as Data deposition: The crystallography, atomic coordinates, and structure factors have been an active ingredient in laundry detergents (6, 7, 9, 10). The ESPs deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2xrm). have proved excellent paradigms for understanding key molecu- 1M.G. and G.K. contributed equally to the work. lar features of proteins, including enzyme catalysis (11) and 2 – Present address: Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig protein folding (6 8). D-04107, Germany. The intracellular subtilisins (ISPs) are a distinctive class found 3To whom correspondence may be addressed. E-mail: [email protected] or keith@ in many different bacilli and related bacteria (1, 12–17). They ysbl.york.ac.uk. are the main component of the bacilli degradome, believed to This article contains supporting information online at www.pnas.org/lookup/suppl/ account for approximately 80% of Bacillus subtilis intracellular doi:10.1073/pnas.1014229108/-/DCSupplemental. 3536–3541 ∣ PNAS ∣ March 1, 2011 ∣ vol. 108 ∣ no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1014229108 Downloaded by guest on October 2, 2021 A Time (hr)(hr) A 0 1234578101 2 3 4 5 7 8 10 U B P 1166 B 100100 14 90 % processed proISP min) / p 12 80 rocessed CD sed y 70 1100 y 60 drol y 8 5050 ctivit p roISP Activity A 6 40 30 4 20 2 10 (µM peptide hydrolysed/min) (µM peptide hydrolysed/min) (µM peptide h Fig. 1. Sequence and structural features of ESPs and ISPs. (A) General 0 0 primary structure of ESP and ISP. SS and Pro refer to the signal sequence 02468100 2 4 6 8 10 and prodomain region, respectively. Black and gray boxes represent the TimeTime (hr)(hr) N- and C-terminal extensions, respectively, of the ISPs. Arrows indicate proteolytic cleavage positions. Approximate positions of the catalytic serine C U resides are indicated by triangles. (B) Partial sequence alignment of the P N-terminal region of ISPs from various Bacillus species and two representative U ESPs (BPN′ from Bacillus amyloliquifaciens and Savinase from Bacillus lentus). P A full sequence alignment has been published previously (21). Residues 013570 1 3 5 7 9 highlighted in black and gray are conserved and semiconserved, respectively. Residues underlined form part of the ESP prodomain sequence. Standard TimeTime ((hr)hr) residue numbering based on mature BPN′ is used for the ESPs. Arrows indi- Fig. 2. Role of proteolytic processing in regulating ISP activity. (A) Time cates the determined proteolytic cleavage position for the ISP from B. clausii course of proISP processing. (B) Change in ISP activity (black squares; left y that is part of this study and the two ESPs. (C) Recently determined structure axis) measured by monitoring the hydrolysis of Suc-Phe-Ala-Ala-Phe-pNA of the dimeric ISP from B. clausii (ref. 21; PDB code 2WV7). Each monomer compared with proISP processing (gray squares; right y axis) as determined is shown in different shades of gray and the catalytic triad as space fill. 250 by densitometry of the bands shown in (A) over time. The rate of proISPS A (D) Binding mode of N-terminal extension. Close up of active site, with the 18 processing by NΔ -ISP (triangle) determined by densitometry of the bands in core ISP structure shown in surface representation and the N-terminal exten- 250 (C) is also shown. (C) Time course of proISPS A processing in the presence of sion shown in stick representation. The positions of residues Pro8, Tyr9, 18 active NΔ -ISP. Top panel, SDS-PAGE. (Lower) Western blot. U and P refer to and Ala250 are indicated. Molecular structure diagrams were generated the unprocessed and processed forms, respectively. using CCP4mg (42). BIOCHEMISTRY Results the processed wt ISP (SEVPM). The rate of processing of S250A Δ18 Proteolytic Processing Regulates ISP Activity. SDS-PAGE revealed proISP by N -ISP followed a standard enzyme progres- proISP was processed to a smaller product after an initial lag sion rate curve compared to the exponential processing charac- B phase in a time dependent manner (Fig.
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