Use of Site-directed Chemical Modification to Study an Essential Lysine in Escherichia coli Leader Peptidase

Mark Paetzel,Natalie C.J Strynadka,William R Tschantz,Ruby Casareno,Patrick R Bullinger,Ross E Dalbey

doi:10.1074/jbc.272.15.9994

Mark Paetzel, Natalie C.J Strynadka + Show 4 more

Open Access

https://doi.org/10.1074/jbc.272.15.9994

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Abstract

Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.

Highlights

Escherichia coli leader peptidase is an integral membrane serine protease that functions to cleave off the aminoterminal leader sequence from proteins that are tar
1 The abbreviations used are: No Cys, the cysteine-less variant of leader peptidase, all native cysteine residues were changed to serine; PAGE, polyacrylamide gel electrophoresis; OmpA, outer membrane protein A; 2BEA, 2-bromoethylamine1⁄7HBr; 3BPA, 3-bromopropylamine1⁄7HBr; 2MEA, 2-mercaptoethylamine; 2BETMA, (2-bromoethyl)trimethylammonium1⁄7Br; DTNB, 5,5Ј-dithiobis(2-nitrobenzoic acid); CAPS, 3-(cyclohexylamino)propanesulfonic acid; TGC buffer, Tris/glycine/CAPS buffer; Me2SO, dimethyl sulfoxide; K145C, No Cys, the leader peptidase mutant containing a unique cysteine at the 145-position; mutation nomenclature used, e.g. K145C, the native lysine at position 145 is changed to cysteine
Purification of Mutants—The use of the 6-His tag/nickel affinity chromatography method [27] of purification has allowed us to purify the overexpressed mutants of leader peptidase away from the wild-type leader peptidase expressed by the E. coli chromosome

Summary

EXPERIMENTAL PROCEDURES

Materials—The 2-bromoethylamine1⁄7HBr (2BEA), 3-bromopropylamine1⁄7HBr (3BPA), (2-bromoethyl)trimethylammonium1⁄7Br (2BETMA), 2-mercaptoethylamine (2-MEA), and (S)-2-aminoethyl-L-cysteine1⁄7HCl were purchased from Sigma. Bacterial Strains and Plasmids—The leader peptidase proteins were expressed in MC1061 E. coli cells harboring the pING plasmid carrying the mutant leader peptidase gene. Six consecutive histidine residues were engineered by oligonucleotide-directed mutagenesis into the P1 (cytoplasmic) domain of leader peptidase that allowed us to purify the mutants from the chromosome-expressed wild-type leader peptidase. The No Cys variant of E. coli leader peptidase was produced by using oligonucleotide-directed mutagenesis to replace the three cysteines at positions 21, 170, and 176 with serine residues. All cloning procedures used T4 kinase, T4 DNA ligase, Klenow, and restriction enzymes from Life Technologies, Inc. Oligonucleotide-directed mutagenesis was performed as described by Zoller and Smith [25]. E. coli MC1061 cells containing the pING plasmid encoding the mutant leader peptidase protein were grown in M9 minimal media (1– 8 liters) containing 100 ␮g/ml ampicillin until an absorbance of 0.5 at 600 nm was reached.

The abbreviations used are

RESULTS

DISCUSSION