title: Substrate Specificity of Signal Peptidase, LepB, as an Adjunct to Design of Novel Inhibitors
Creators: name: Musik, Joanna
Publisher: name: Griffith University
Year: 2022
Subjects: subject: signal peptidase
subject: Design of Novel Inhibitors
subject: LepB

description: Bacterial secreted proteins require an N-terminal signal peptide to leave the cytoplasm. Once the protein has been translocated across the cytoplasmic membrane, the signal peptide is cleaved by a signal peptidase (SPase), allowing the remainder of the protein to fold into its mature state. SPase I found in prokaryotes (P-type) and eukaryotes (ER-type) have the same function, but are structurally different, which makes bacterial SPase I an attractive antibiotic target. The major difference between the P-type and ER-type SPases is the catalytic dyad present in the active site, with prokaryotes utilising a Ser-Lys dyad, while eukaryotes utilise a Ser-His dyad. SPase I of E. coli, LepB, removes the signal peptide from non-lipoproteins and cleaves after the canonical sequence Ala-X-Ala. Amino acids surrounding the SPase I cleavage site have been shown to affect the efficiency of protein secretion. The amino acids before the cleavage site (P1 onwards) have been well studied. However, the amino acids after the cleavage site, in the mature protein region (P1′ onwards), have not been not been studied thoroughly and are the focus of my PhD studies. We first investigated a bias against aromatic amino acids at the second position after the cleavage site (P2′) in E. coli secreted proteins. Maltose binding protein (MBP) was mutated to introduce aromatic amino acids (tryptophan, tyrosine and phenylalanine) at P2′. All mutants with aromatic amino acids at P2′ were exported less efficiently, indicated by an increase in precursor protein. The binding kinetics of peptides that encompass the MBP cleavage site to LepB also showed a slower off-rate compared to MBP wild-type peptide. A study of secreted proteins to look for aromatic amino acid at P2′, revealed the Bacillus subtilis protein TasA, which contains a phenylalanine at P2′. B. subtilis has five type I signal peptidases, one of these, SipW, is an ER-type peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an ER-type signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by LepB. Expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2′) to both maltose binding protein (MBP) and β- lactamase (Bla); both model systems of E. coli secretion. We observed a defect in secretion, indicated by accumulation of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of point mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around II the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2′, inhibit processing by LepB. To determine why the TasA sequence was inhibitory in E. coli, we designed 11 peptides to mimic the inefficiently cleaved secreted proteins, wild-type TasA and the TasA-MBP fusion. These peptides were studied to determine whether the peptides interact with and inhibit the function of LepB. The binding affinity and inhibitory potential of the peptides against LepB was assessed by surface plasmon resonance (SPR) and a LepB enzyme activity assay. Molecular modelling of the interaction between TasA-MBP and LepB indicated that the tryptophan residue at P2 (two amino acids before the cleavage site) inhibited the active site serine-90 residue on LepB from accessing the cleavage site. Substituting the P2 tryptophan with alanine (W26A) allowed for more efficient processing of the signal peptide when TasA-MBP was expressed in E. coli. In this study we determined that aromatic amino acids at P2′ of secreted proteins leads to a slower release from SPase I. In addition, we uncovered the potential of the TasA signal peptide and early mature region to be used as an inhibitor for P-type SPases as it is cleaved by the ER-type SPase SipW, but inefficiently cleaved by the P-type SPase LepB.