Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.


A S W A A G A A I E G R A A G A A E C N
) fused to the NisA-leader and produced with or without NisBC co-expression.
The protease resistance relative to untreated (no FXa) samples was calculated and data representing mean ± s.d. of three independent phage preparations analyzed in duplicate is shown (unpaired, twotailed t-test). (c) As in (b), but phage displayed model peptides containing ProcA leader sequences (full sequences in Supplementary Table 4) were tested after phage production with or without ProcM co-expression. The experiments shown in panel b and c were repeated twice. a Display NisA-precursor peptides Display ProcA-precursor peptides uPA incubated with ProcM-modified anti-uPA leader-core peptides PEP332 to PEP335 as indicated.
(b) As in (a), but residual activities of human uPA incubated with PEP333 produced with (+ ProcM) or without (-ProcM) ProcM co-expression. The inhibitory constant Ki of PEP333 is 564 nM and was calculated as recently described 2 . The experiment was repeated three times.  The N-terminal sequence shared by all constructs is comprised of the OmpA signal sequence (OmpA residues 1-21) fused to a linker sequence containing an (HK)3-repeat for improved expression and the natural NisA-leader sequence (underlined); XXX indicates core peptide sequences as shown in the individual rows below. b Plasmid trivial name used in this study. c Core peptide sequence with FLAG-and His6-tags used for ELISA capture/detection (shaded), FXa-site (bold, underlined), and relevant residues for thioether-bridge formation (double-underlined). Supplementary Table 4 Amino acid sequence of ProcA-leader containing model lanthi-precursor peptides displayed on phage pIII CT and expressed from pL3_stuffer, or of pL3C_zeoR-stuffer derivatives OmpA

Supplementary
a Model lanthi-precursor peptide fusion to the N-terminus of pIII is comprised of the OmpA signal sequence (OmpA residues 1-21), a short linker, the ProcA3.3-leader (underlined), the core sequence (indicated by XXX; sequence in the row below), a linker, and residues 275-424 of phage pIII. b The sequence shared by all lanthi-precursor peptides fused to the C-terminus of pIII is comprised of the OmpA signal sequence (OmpA residues 1-21), residues 275-424 of phage pIII, a linker sequence, and the ProcA3.3-leader (underlined); XXX indicates core peptide sequences as shown in the individual rows below. c Plasmid trivial name used in this study. d Core peptide sequence with HA-and His6-tags (shaded), FXa-site (bold, underlined), and relevant residues for thioetherbridge formation (double-underlined).

Amino acid sequence of antimicrobial lantibiotic precursor peptides fused to the C-terminus of MBP and expressed from pET21a_MBP_FXa_zeoR-stuffer derivatives
HHHHHHGTMSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCHC SIHVSK e a The N-terminal sequence shared by all constructs, with the exception of PEP226, is comprised of MBP residues 27-391 (Met start codon, no signal sequence) fused to a linker sequence containing an internal FXa-site, and the natural ProcA3.3leader sequence (underlined); in PEP226 the linker sequence was replaced by NSSSNNNNNNNNNNGTPGGSGGAPGS to eliminate the FXa-site; XXX indicates core peptide sequences as shown in the individual rows below. b Plasmid trivial name used in this study. c Core peptide sequence with His6-tag (shaded). d The N-terminal sequence shared by the two lantibiotic fusions is comprised of MBP residues 27-391 (Met start codon, no signal sequence) fused to a linker sequence containing an internal FXa-site; XXX indicates peptide precursor sequences as shown in the individual rows below. e Precursor sequences consisting of the ProcA3.3 and NisA leader sequences (underlined), the LctA and NisA core peptides (bold), and N-terminal His6-tag (shaded).  ). b Calculated mass of singly (zn 1+ ), doubly (zn 2+ ) and triply (zn 3+ ) charged c-fragments. Xaa-Pro sites do not give rise to fragments under ETD conditions due to the pyrrolidine structure of the proline side chain. Sequences comprising the proposed thioether ring are highlighted (gray). Peptide sequences are shown without the N-terminal ProcA3.3 leader (GSPGGSGGAPGSMSEEQLKAFIAKVQGDSSLQEQLKAEGADVVAIAKAAGFTIKQQDLNAAASELSDEE-LEAASGG) and the C-terminal His6-tag (AGPHHHHHH). Putative sites of dehydration are underlined. c Modifications found are -nx H2O; dehydration of Ser and Thr (-H2O; -18.01057 Da), P; phosphorylation (+HPO3 ; +79.96633 Da), SS; disulfide formation (-2H; -2.01565 Da) and GSH; glutathione addition (+ 307.083801 Da). d The height of the isotopic mass distribution was used to estimate the percentage of each fraction when multiple species are detected. e PEP333u is peptide produced in absence of ProcM enzyme (u = unmodified) and was used as internal positive control for the iodoacetamide modification, since it has two free cysteine residues. f SAP stands for treatment with shrimp alkaline phosphatase and only sample PEP330 was determined to demonstrate phosphorylation (n.d. is not determined). g TCEP was used to reduce the peptide samples to demonstrate the presence of disulfide bridge. h IAA stands for iodoacetamide modification of cysteine (+C2H3NO; + 57.02146 Da). The number of additions and their relative appearance based on isotopic mass distribution peak height is shown. Each reaction tube included PEP333u as a positive control for full IAA modification. i Zero lanthionines is indicated here since these peptide species are disulfide-bridged and contain a free dehydroalanine, which reacts promptly with free cysteine at pH 8. -----a CID (Collision-induced dissociation). The residues highlighted in gray are part of the proposed lanthionine ring. b Due to the presence of 4 Arg residues in the sequence these masses can only be observed with loss of two ammonia. c Dehydrated residue; Dhb (dehydrobutyrine), Dha (dehydroalanine). d A very clear related c13 1+ -ion peak was detected (found m/z 1312.578 and calc. m/z 1312.579).  116.071 --a CID (Collision-induced dissociation). The residues highlighted in gray are part of the proposed lanthionine ring. b Dehydrated residue; Dhb (dehydrobutyrine), Dha (dehydroalanine). ------29 H 1 156.077 78.542 a CID (Collision-induced dissociation). The residues highlighted in gray are part of the proposed lanthionine ring. b Dehydrated residue; Dhb (dehydrobutyrine), Dha (dehydroalanine).  .102 --a CID (Collision-induced dissociation). The residues highlighted in gray are part of the proposed lanthionine ring. b Dehydrated residue; Dhb (dehydrobutyrine), Dha (dehydroalanine). c The triple charged phosphorylated species was used to generate these MS/MS data. Phosphorylation is not observed therefore one of these dehydrated residues results from MS/MS induced dephosphorylation.

Bioactivity assay
Sequences encoding the NisA precursor from L. lactis or a chimera consisting of the ProcA3.3 leader peptide fused to the LctA core peptide 1 were fused to the MBP gene via a linker that encodes an FXa cleavage site followed by a His 6 -tag, resulting in plasmids pET21a_MBP_His 6 _NisA and pET21a_MBP_His 6 _ProcA-LctA, respectively. Heterologous production of MBP-fusions in E. coli and purification of leader-core peptides with N-terminal His 6 -tag was performed as described above. In brief, pET21a_MBP_FXa_NisA was produced with and without co-expression of the NisBC enzymes, whereas pET21a_MBP_FXa_ProcA-LctA was produced with and without co-expression of the ProcM enzyme. Following purification of the MBP-fusions by Dextrin-Sepharose affinity chromatography and FXa-digest, the released His 6 -tagged leader-core peptides were purified by IMAC and adjusted to a final concentration of 20 µM in PBS. Core peptides were released by treatment with 5 µM Trypsin (Sigma) for 3 h at 37°C followed by 10 h at 30°C and heatinactivation at 99°C for 5 min. 40 µl of the resulting peptide solutions were directly spotted onto filter discs placed on solidified top-agar solutions containing appropriate indicator strain bacteria. Trypsin-treated PBS served as negative control. To assess the bioactivity of NisA (derived from pET21a_MBP_His 6 _NisA production) a stationary phase L. lactis NZ9000 culture grown in GM17 media was diluted 1:25 into 15 ml GM17 top-agar and poured onto a 30 ml layer of bacteria-free GM17 agar. Authentic nisin was obtained from the cell-free supernatant of an L. lactis NZ9700 overnight culture grown in G17 media and served as positive control. To assess the bioactivity of the ProcA-LctA chimera (derived from pET21a_MBP_ His 6 _ProcA-LctA production) a stationary phase L. lactis subsp. cremonis

Determination of inhibitory activity of lanthipeptides
The inhibitory activities of ProcM-modified and non-modified anti-uPA peptides were determined as previously described 2 . In brief, 1.