Abstract
Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.
Highlights
Proteins destined for secretion are synthesized in a precursor form with an amino-terminal extension peptide that targets the exported protein to the Sec machinery [1] or the Tat machinery [2] in bacteria
We have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the ؊1 position
A critical serine and lysine residue is present in SPases from other species of bacteria [12], and members of the signal peptidase I family in mitochondria [13]
Summary
Bacterial Strains and Plasmids—The E. coli strain DH5␣ was obtained from our laboratory collection the E. coli temperature-sensitive SPase I strain, IT41, was obtained from Dr Yoshikazu Nakamura [21]. A plasmid capable of expressing E. coli TEM-1 -lactamase (UniRef90_P62593) was needed that could be simultaneously transformed with the SPase mutants For this vector, we modified pRD8 by removing the SPase gene [23]. The plasmid, pUC19, contains the pMB1 origin of replication and can be co-transformed with the pGZ119HESPase constructs It expresses -lactamase constitutively at high level. -Lactamase fractions purified by phenylboronate-agarose chromatography were first precipitated with a final concentration of 12% trichloroacetic acid for 1 h on ice. Precipitated proteins were centrifuged at maximum speed at 4 °C in an Eppendorf microcentrifuge for 20 min. The mass accuracy in the 30-kDa mass range is approximately Ϯ25 Da. Pulse-Chase Assay of -Lactamase Processing—Competent cells of the IT41 strain were prepared using the CaCl2 method and co-transformed with the various mutants of the pGZ119HE-SPase and pING vectors. The surface and binding site analysis was performed using the CASTp server [33]
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