The Type 4 Prepilin Peptidases Comprise a Novel Family of Aspartic Acid Proteases*
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 2, Issue of January 14, pp. 1502–1510, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Type 4 Prepilin Peptidases Comprise a Novel Family of Aspartic Acid Proteases* (Received for publication, August 13, 1999, and in revised form, October 29, 1999) Christian F. LaPointe‡ and Ronald K. Taylor§ From the Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755 Type 4 prepilins or prepilin-like-proteins are secreted The related type 4 pilin-like proteins partially comprise the by a wide range of bacterial species and are required for main terminal branch of the general secretory pathway in a variety of functions including type 4 pilus formation, Gram-negative bacteria, also known as the type 2 secretion toxin and other enzyme secretion, gene transfer, and pathway (6). The general secretory pathway is the pathway biofilm formation. A distinctive feature of these proteins utilized by the majority of proteins that are secreted outside of is the presence of a specialized leader peptide that is Gram-negative bacteria. Such proteins include toxins such as cleaved off by a cognate membrane-bound type 4 prepi- cholera toxin of V. cholerae (7) and exotoxin A of Pseudomonas lin peptidase (TFPP) during the process of secretion. In aeruginosa (8), as well as other enzymes such as pullulanase of this report we show that the TFPPs represent a novel Klebsiella pneumoniae (9) and hemolysin of Aeromonas hy- family of bilobed aspartate proteases that is unlike any drophila (10). Type 4 pilin-like proteins are also required for other protease. The active site pairs of aspartic acids of the two TFPPs in Vibrio cholerae are found at positions natural transformation of Bacillus subtilus (11) and Strepto- 125 and 189 of TcpJ and 147 and 212 of VcpD. Corre- coccus pneumoniae (12). sponding aspartate residues are completely conserved The type 4 leader peptide is characterized by several features throughout this extensive peptidase family. that are highly conserved, yet differ from those of standard leader peptides. The N-terminal leader that is cleaved from the mature protein is highly charged and immediately precedes a Members of the family of leader peptidases known as type 4 region of approximately 20 residues that are predominantly prepilin peptidases (TFPP)1 have been identified in a vast hydrophobic and are retained within the mature protein. The number of species of Gram-negative bacteria and an increasing majority of type 4 prepilins or prepilin-like proteins are desig- number of Gram-positive bacteria. The TFPP is responsible for nated type 4a, containing leader peptides that are short (ϳ6 the cleavage and N-methylation (known collectively as process- residues). Type 4b preproteins contain a longer leader (ϳ25 ing) of the highly conserved type 4 leader peptide present at the residues). The peptide bond on the prepilin that is hydrolyzed N terminus of the families of secreted proteins known as type 4 by the TFPP lies between the charged and hydrophobic do- prepilins and type 4 prepilin-like proteins (1). Whereas the mains, immediately C-terminal to an invariant glycine. The cleavage of the type 4 leader peptide is a necessary step for new N-terminal amino acid that arises upon processing is a secretion of the mature pilin, methylation of the N terminus of N-methylated phenylalanine for type 4a proteins and is typi- the mature pilin is not required for secretion and has no known cally a N-methylated methionine for type 4b proteins. The function (2). Type 4 pilins are polymerized as the structural TFPPs themselves are integral cytoplasmic membrane proteins subunits of type 4 pili (Tfp), which are surface organelles re- with numerous transmembrane domains. The processing reac- quired for diverse activities that contribute to broad attributes tion has been postulated to occur on the cytoplasmic side of the such as genetic transfer, virulence, and environmental persist- membrane because of the membrane orientation of the prepi- ence of a wide array of Gram-negative bacterial pathogens. lin, the location of all the major nonmembrane peptidase do- Prototypical examples include microcolony formation mediated mains, and the likelihood that the cytoplasmically located S- by toxin-coregulated pilus (TCP) of Vibrio cholerae,2 bacterial adenosyl methionine would provide an available source of surface dispersal mechanisms mediated by bundle forming pi- methyl groups for the methylation step of the reaction (13). lus of enteropathogenic Escherichia coli (3), colonization and Previous studies on the proteolytic mechanism of the TFPPs natural transformation mediated by PilE of Neisseria sp. (4), have focused on the largest cytoplasmic region of the protein and gliding motility mediated by the Tfp of Myxococcus sp. (5). designated cytoplasmic domain 1. These studies were per- formed on PilD, a prototypic TFPP from P. aeruginosa. Domain * This work was supported in part by Grant ROI AI 23096 from the 1 of the PilD peptidase contains two pairs of cysteine residues National Institutes of Health. The costs of publication of this article that are conserved among many members of the TFPP family. were defrayed in part by the payment of page charges. This article must Alteration of the 4 highly conserved cysteine residues by sub- therefore be hereby marked “advertisement” in accordance with 18 stitution of alanine or serine residues resulted in up to an U.S.C. Section 1734 solely to indicate this fact. ‡ Recipient of Predoctoral Fellowship AI F31-09635 from the Na- 80–100% reduction of peptidase activity in an in vitro assay. tional Institutes of Health. However, the processing defect varied from approximately 0% § To whom correspondence should be addressed: Dept. of Microbiol- to 80% for the various mutants in vivo, suggesting that al- ogy, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650- though pilin cleavage was less efficient, it was not abolished 1632; Fax: 603-650-1318; E-mail: [email protected]. 1 The abbreviations used are: TFPP, type 4 prepilin peptidase; Tfp, (14). Chemical inhibitor studies supported the notion that the type 4 pili; TCP, toxin-coregulated pilus; NEM, N-ethylmaleimide; cysteine residues contributed to protease activity and PilD was PCR, polymerase chain reaction; Ab, antibody; PAGE, polyacrylamide classified as a cysteine protease, specifically as a member of the gel electrophoresis; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi- C20 family of cysteine proteases (15). The retention of partial imide hydrochloride. 2 Kirn, T. J., Lafferty, M. L., Sandoe, C. M. P., and Taylor, R. K. activity by some of the pilD mutants as well as the subsequent (2000), Mol. Microbiol., in press. discovery of TFPP’s that lack the consensus cysteine residues 1502 This paper is available on line at http://www.jbc.org Protease Active Site of TFPP 1503 have brought this mechanism of protease activity into question. TcpA Prepilin—The method for membrane preparation was adapted For example, the XpsO TFPP of Xanthomonas campestris was from a previously described cell fractionation protocol (25). A 100-ml characterized and shown to lack all the domain 1 cysteine overnight culture was placed on ice for 20 min, and the cells were then collected by centrifugation at 5000 ϫ g for 10 min. The cell pellet was residues, yet the xpsO gene fully complements a pilD deletion resuspended in 1 ml of 200 mM Tris, pH 8.0, to which 1 ml of 50 mM Tris, for prepilin processing (16). Some newer members of the rap- pH 8.0, 1 M sucrose, 2 ml of H2O, 20 lof0.5M EDTA, and 20 lof idly growing family lack the entire domain 1 region. Analysis of lysozyme (10 mg/ml) were added. The cell suspension was allowed to a current TFPP alignment also reveals a complete lack of any incubate on ice for an additional 30 min. 100 lof1M MgSO4 was conserved histidine residue, a necessary component of the cys- added, and the suspension was centrifuged at 5000 ϫ g for 10 min. The ϫ teine protease catalytic mechanism (15). These observations cell pellet is resuspended in 5 ml of 50 mM Tris, pH 8.0, sonicated 3 15 s at 50% duty, and centrifuged at 5000 ϫ g for 10 min. The super- have led to a revised classification of PilD, and therefore the natant was then centrifuged at 230,000 ϫ g (60,000 rpm in a TLA 100.3 complete family of TFPPs, to the U12 category of proteases rotor) for 15 min. The resulting pellet, which contained the total mem- with unknown catalytic mechanism (17). brane fraction, was resuspended in 200 lof50mM Tris, pH 8.0. In the study reported here, an approach similar to the com- TcpA Prepilin Quantitation—Membrane preparations were prepared prehensive mutational strategy employed to identify the signal from 42 °C grown overnight cultures of E. coli K38, which does not peptidase I active site (18) was used to determine the active site express TcpA, and MK90, which overexpresses TcpA prepilin at ele- vated temperature. 20 l of a membrane preparation from K38 and residues of TcpJ, the TFPP responsible for processing the TcpA from MK90 were subjected to electrophoresis on a 12.5% SDS-PAGE gel type 4 prepilin of V. cholerae. A protein sequence alignment of alongside a standard of 3 g of trypsin, followed by staining with the TFPP family revealed several highly conserved potential Coomassie Blue. The stained gel was dried using the Novex gel drying protease active site residues in addition to the two cysteine system. The dried gel was scanned by a Molecular Dynamics Personal pairs that exist in this protease family. These include serine, Densitometer SI, and densitometry analysis was performed using Im- aspartic acid, lysine, and glutamic acid residues, most of which ageQuant version 1.2 software.