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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.

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.

International audience; LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.