Altered -3 Substrate Specificity of Escherichia coli Signal Peptidase 1 Mutants as Revealed by Screening a Combinatorial Peptide Library

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, 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. During the export process, the signal peptide is cleaved from the precursor protein by a signal peptidase that is embedded in the plasma membrane.AIn Escherichia coli, signal peptidase (SPase I) 2 consists of a single polypeptide chain of 37 kDa (3). This enzyme spans the membrane twice with a small cytoplasmic segment (residues 29 -58) and a large carboxyl-terminal catalytic domain located in the periplasm (residues 77-323) (4 -6). Catalysis by SPase I is carried out by a Ser-Lys dyad (7-10). In the case of the E. coli SPase I, Ser-90 is the nucleophilic residue that attacks the scissile bond of the precursor substrate and lysine 145 is the general base that deprotonates the serine residue (for review, see Ref. 11). A critical serine and lysine residue is also present in SPases from other species of bacteria (12), and members of the signal peptidase I family in mitochondria (13).With the exception of the mitochondrial inner membrane peptidase I (Imp1), all type I signal peptidases carry out processing with a specificity for small aliphatic residues at the Ϫ1 (P1) and Ϫ3 (P3) positions (11). Alanine is usually the preferred amino acid residue at the Ϫ1 and Ϫ3 positions and results in the frequently observed "Ala-X-Ala" motif for signal peptide cleavage (14 -16). The residues of SPase I that comprise the substrate binding site have been identified by solving the x-ray structure of the soluble catalytic domain with a covalently attached 5S penem inhibitor (10) and a structure with a non-covalent lipohexapeptide inhibitor (17). The three-dimensional structure of SPase I with no inhibitor bound (apo-structure) revealed that there is some variation in the binding pocket volume when compared with the inhibitor-bound structures (18). The E. coli SPase residues making direct van der Waals contact with the P1 methyl group are . Those making contact with the P3 residues are Phe-84, Ile-144, Val-132, and Ile-86. The substrate binding to SPase I occurs in an extended conformation. Recently, we have made mutations of the E. coli SPase I in the S1 and S3 pockets that bind the P1 and P3 residues of the substrate to identify the residues that control the substrate specificity (19). We found that alterations of the Ile-144 and Ile-86 residues to alanine residues could alter the substrate specificity and lead to cleava...

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

“…One possible explanation for the lack of fidelity is that the cysteine mutation in the binding site results in sliding of the substrate within the active site such that alternative peptide bonds can be hydrolyzed. Moreover, Ile144 and Ile86 control substrate specificity, because mutation of these residues results in substrate cleavage even with an arginine at the Ϫ3 position (35).…”

A Small Subset of Signal Peptidase Residues are Perturbed by Signal Peptide Binding

“…The residues with altered chemical shifts were mapped onto the crystal structure of the SPase I Δ2–75 (Figure 3) (5). Virtually all of the perturbed residues are located in one groove on the surface of the protein (Figure 3A).…”

“…A model of a wild-type alkaline phosphatase signal peptide cleavage region plus the C-terminal most core region Phe residue (Phe-Cys-Pro-Val-Thr-Lys-Ala) bound to the binding site of SPase I Δ2–75 was prepared using the atomic coordinates from the model of the SPase I Δ2–75 complex with the cleavage region of the beta-lactamase signal peptide deduced from molecular modeling and generously provided by Mark Paetzel (5). Backbone-dependent changes in the signal peptide sequence of the model were prepared using the PyMOL Viewer Software.…”

“…The enzyme is thought to catalyze peptide bond hydrolysis via a catalytic dyad involving the hydroxyl of Ser90 for nucleophilic attack and the ε-amine of Lys145 as the general base to cleave the preprotein. Crystal structures of the soluble catalytic domain (SPase I Δ2–75) in the apo-form (3) and with bound inhibitors (4,5) revealed two shallow hydrophobic cavities adjacent to the Ser–Lys dyad that would be appropriate for docking residues −1 and −3 of the signal peptide.…”

“…To date, these interactions have been inferred from co-crystals of SPase I Δ2–75 with 5 S , 6 S β-lactam (penem) (4) or with the fatty acid derivative, Arylomycin A2 (5), both inhibitors of SPase I activity, and from molecular modeling using the β-lactamase signal peptide to predict its binding locale (5). However, no direct structural analysis of the enzyme with bound signal peptide or preprotein has been accomplished.…”