Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library.
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.
Ekici
Ozlem Dogan
OD
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Karla
Andrew
A
Paetzel
Mark
M
Lively
Mark O
MO
Pei
Dehua
D
Dalbey
Ross E
RE
D001216:Asparagine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D015723:Gene Library; D007532:Isoleucine / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000235:genetics*; D013379:Substrate Specificity
D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D007532:Isoleucine; D010649:Phenylalanine; D001216:Asparagine; D012697:Serine Endopeptidases; C026515:type I signal peptidase