Versatility of Prolyl Oligopeptidase B in Peptide Macrocyclization

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Versatility of prolyl oligopeptidase B in peptide macrocyclization Robert Michael Sgambelluri, Miranda O Smith, and Jonathan D Walton ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00264 • Publication Date (Web): 02 Sep 2017 Downloaded from http://pubs.acs.org on September 4, 2017

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ACS Synthetic Biology

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Versatility of prolyl oligopeptidase B in peptide macrocyclization R. Michael Sgambelluri1,2, Miranda O. Smith2, and Jonathan D. Walton2,3,* 1

Department of Biochemistry and Molecular Biology, 2Department of Energy-Plant Research

Laboratory, and 3Department of Plant Biology, Michigan State University, E. Lansing MI 48824 USA

ABSTRACT: Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35-mer precursor peptides which are ultimately converted to hydroxylated bicyclic octapeptides. The initial cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of the 10-amino acid leader sequence from the precursor peptide and transpeptidation to produce a monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide cyclization was systematically characterized using a range of precursor peptide substrates produced either in E. coli or chemically. Substrates produced in E. coli were expressed either individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide substrates were tested, of which POPB could cyclize 100. Peptides of 7 to 16 residues were cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified amino acids including D-Ala, β-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial cyclization was seen when L-Thr at position #5 was replaced with the naturally occurring D amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.

Keywords: amanitin, prolyl oligopeptidase, cyclic peptide, cycloamanide, Amanita

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Due to their structural rigidity and conformational diversities, cyclic peptides often display high affinity binding to target macromolecules, relatively high membrane permeability, and resistance to proteases.1-4 Nine cyclic peptide drugs have been approved in the past ten years against bacterial and fungal infections, cancer, and gastrointestinal disorders.5 Recent examples of promising cyclic peptide drug leads include an inhibitor of the RAS oncogene;6 the modified griselimycins, which have promise against multi-drug resistant tuberculosis;7 a cyclotide that activates the p53 tumor suppressor pathway;8 and lugdunin, a novel antibiotic from a human commensal bacterium that is active against Staphylococcus aureus.9 However, synthesis of cyclic peptides remains difficult and expensive compared to linear peptides.10 Ribosomally biosynthesized cyclic peptides, known as RiPPs, have been described from bacteria, plants, mammals, and fungi.11 Prior to the discovery of the genes encoding the amatoxins, phallotoxins, and other cyclic peptides from the agaric genus Amanita (collectively known as the cycloamanides) RiPPs were unknown in fungi.12-14 Amatoxins such as α-amanitin are defining inhibitors of RNA polymerase II, and phallotoxins such as phalloidin bind and stabilize F-actin.15-17 The amatoxins are highly stable and rapidly absorbed into the bloodstream and into mammalian cells.18 Cycloamanides are biosynthesized initially as small (33-37-amino acid) precursor peptides encoded by a gene family comprising ~35 members in different Amanita species.12,19,20 The conserved structures of the cycloamanide precursor peptides are composed of a 10-amino acid leader, a variable region of 6-10 amino acids which give rise to the mature toxins, and a conserved follower peptide of 17 residues. Although the amino acid content of the variable region in the naturally occurring cycloamanide gene family is biased towards hydrophobic amino acids and especially Pro, all 20 amino acids are present in at least one predicted cycloamanide.20 Cyclization of the variable region of the cycloamanides occurs in two nonprocessive steps, both catalyzed by a specialized prolyl oligopeptidase, POPB (Figure 1).21 The amatoxins and phallotoxins, but not the classic monocyclic cycloamanides, are further posttranslationally processed by multiple hydroxylations and formation of a cross-bridge between Cys and Trp called tryptathionine.22 Additional modifications include sulfoxidation in the amatoxins and epimerization of one amino acid in the phallotoxins.18


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Figure 1. Two-step nonprocessive reaction catalyzed by POPB from Galerina marginata with the α-amanitin precursor peptide as substrate. Adapted from ref. 21.

The kinetic efficiency of POPB from Galerina marginata expressed in Saccharomyces cerevisiae (not G. marginata itself)23, is sufficiently high to make it a practical reagent for custom synthesis of cyclic peptides. POPB is comparable in catalytic properties to the peptide macrocyclase butelase 1 from Clitoria ternatea and PCY1 from Saponaria vaccaria.23-26 In regard to the structural requirements of the precursor peptide substrate for recognition by POPB, the length and composition of the 10-amino acid leader were shown to be important for the initial hydrolytic cleavage, but a leaderless 25mer is cyclized as efficiently as the full-length peptide.21 In the follower peptide, length and secondary α-helical structure are important but primary sequence is not, i.e., the α-amanitin precursor peptides from A. bisporigera and G. marginata are both 35 amino acids in length but their follower peptides are only 24% identical.27 The ultimate Cys residue of the precursor peptide is highly conserved in the cycloamanide superfamily in Amanita and Galerina and changing it to Ala blocks processing by POPB.20,21 Detailed kinetic studies on POPB expressed in E. coli confirmed its high catalytic efficiency as a peptide macrocyclase and showed that release of the follower peptide is the limiting step.23 Here we explore the utility of POPB as a general catalyst for peptide macrocyclization through characterization of the enzyme’s substrate versatility and limitations on composition and length of the core domain sequence.



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RESULTS AND DISCUSSION

1.1. Enzyme and Substrate Preparation. Recombinant POPB enzyme from Galerina marginata was produced in yeast with an N-terminal myc epitope tag21 and purified on anti-cmyc agarose followed by anion exchange (Supplemental Figure S1). As a source of precursor peptide substrates, a strategy was developed for their expression in E. coli. The coding sequence for the amanitin precursor peptide (AMA1) from G. marginata was expressed as a maltosebinding protein (MBP) fusion by cloning into the vector pMAL-c5x (Figure 2). This afforded high stability, yields, and tractability of the precursor peptides. After induction of expression, the MBP fusion proteins were purified from cell extracts on amylose resin. Treatment with Factor Xa protease released the GmAMA1 peptide from the C-terminus of MBP (Figure 2), and the MBP was then removed by precipitation with methanol. LC/MS indicated the release of GmAMA1 from the fusion protein upon treatment with Factor Xa and formation of cyclo(IWGIGCNP) upon addition of GmPOPB enzyme (Figure 2). Approximately 6 mg of precursor peptide were produced from one liter of bacterial culture.

Figure 2. (A) SDS-PAGE of AMA1 precursor peptide expression and purification as a maltose binding protein (MBP) fusion. (B) LC/MS indicating release of the AMA1 peptide by Factor Xa protease and formation of cyclo(IWGIGCNP) by POPB.


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1.2. Amino Acid Preferences for Cyclization. Site-directed mutagenesis of the wild-type AMA1 expression construct was used to generate a series of mutants with amino acid substitutions at each position, excluding Pro8, which was presumed to be essential for POPB recognition. Reactions typically contained ~5 µg POPB, ~25 µg substrate, and ran for 4 hr at 37°C. The results are summarized in Table 1 and the corresponding LC/MS chromatograms are shown in Figs. S2-S8. Cyclic products were produced from all 28 substrates. All substitutions in

Table 1.Tolerance of POPB for amino acid substitutions in the core region, whose wild type sequence is Ile1-Trp2-Gly3-Ile4-Gly5-Cys6-Asn7-Pro8. Wild type sequences are coded green, reactions that did not yield >90% cyclic product are coded pink. Corresponding LC/MS traces are shown in Supplemental Figures S2-S8.



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Position

Amino Acid Type

Residue

Reaction Progress (%) a

Cyclic (%)b

1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 7 7 7 7 7

wild type small, nonpolar small, polar large, nonpolar large, polar wild type small, nonpolar small, polar large, nonpolar large, polar wild type small, nonpolar small, polar large, nonpolar large, polar wild type small, nonpolar small, polar large, nonpolar large, polar wild type small, nonpolar small, polar large, nonpolar large, polar wild type small, nonpolar small, polar large, nonpolar large, polar wild type small, nonpolar small, polar large, nonpolar large, polar

Ile Ala Ser Leu Asn Trp Ala Ser Phe Asn Gly Ala Ser Leu Asn Ile Ala Ser Leu Asn Gly Ala Ser Leu Asn Cys Ala Ser Leu Asn Asn Ala Ser Leu Gln

> 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 72 33 85 > 99 > 99 > 99 > 99 > 99 > 99 > 99 88 92 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99 > 99

> 99 > 99 > 99 > 99 > 99 > 99 > 99 32 > 99 46 > 99 > 99 76 18 63 > 99 > 99 > 99 > 99 > 99 > 99 > 99 74 60 64 > 99 > 99 > 99 98 97 > 99 > 99 > 99 > 99 > 99

Footnotes: a

Reaction progress indicates substrate consumption over 4 hr.

b

Cyclic % indicates percent of product in the cyclic vs. linear form after 4 hr.


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residues #1, #4, #6, and #7 gave yields of >99% of cyclized core domain. Some substitutions at positions #2, #3, and #5 were less well tolerated and gave higher yields of linear octamer product resulting from preference of the enzyme for hydrolysis over transpeptidation in the second catalytic step. Decreased yields were observed when residue #2 was changed to the polar amino acids Ser or Asn, suggesting a preference for nonpolar residues at this position. POPB tolerated Ala but not Ser, Leu, or Asn, at positions #3 and #5. The cyclic product yields for these lesspreferred substrates ranged from 18% (G3L) to 76% (G3S). While the incubation time used in these assays was intended to allow the reactions to run to completion, detectable amounts of full-length substrate remained in the assays with five of the mutants (G3S, G3L, G3N, G5S, G5L), all of which also gave reduced yields of cyclic product. To test whether the reduced cyclization efficiency was due to reduced first-stage hydrolysis, 25mer forms (i.e., without the 10-amino acid leader) of four of the substrates (wild-type, W2S, G3L, and G5S) were tested as substrates for POPB. POPB cyclase activity is as efficient with the 25mer as with the native 35mer.21 The same efficiencies in cyclization were observed with the 25mer substrates (Table S1). Thus, these substitutions (W2S, G3L, and G5S) resulted in poorer substrates for both hydrolysis and cyclization steps. 1.3. Cyclization of Sequences Containing Unusual Amino Acids. Amatoxins and phallotoxins contain up to four hydroxylations. Both groups of toxins have 4-hydroxy-Pro, which is critical for high affinity binding of α-amanitin to pol II.17,18 The amatoxins also contain 6hydroxy-Trp, which is the preferred site for attachment of antibodies in antibody-amanitin conjugates targeted against cancer cells.28,29 It is not known whether the hydroxylations occur before or after cyclization by POPB. In either case, cyclizing the amanitin precursor with the Pro and Trp hydroxylations already in place would facilitate progress towards the complete in vitro biosynthesis of α-amanitin, which to date has eluded chemical synthesis. Furthermore, the compatibility of POPB with unusual amino acids such N-methylated amino acids and/or β-amino acids would expand the utility of POPB to make novel cyclic peptides. We chemically synthesized four additional substrates that contained the modified amino acids trans-4-hydroxy-Pro, 5-hydroxy-Trp, N-methyl-Ala, and β-Ala. (an Fmoc derivative of 6hydroxy-Trp was not commercially available). These substrates were prepared as the 25mer forms lacking the N-terminal leader domain.



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All four of these substrates were cyclized by POPB (Figure 3). Reduced yields were observed from the substrate containing N-methyl-Ala, which gave primarily linearized product. After 4 hr, 26% of the 4-hydroxy-Pro substrate remained, indicating that both hydrolysis and transpeptidation of this substrate was less efficient than other substrates. POP enzymes achieve Pro specificity through a ring stacking interaction between Pro and an active site Trp,30 and this interaction might have been adversely affected by the hydroxyl group. The results indicate that POPB can tolerate amino acids beyond the proteinogenic twenty.

Figure 3. Cyclization of peptides containing unusual amino acids. The modified residues are highlighted in red. Synthetic linear 25mers were incubated with POPB for 4 hr and the reactions analyzed by LC/MS. Shown are overlaid extracted ion chromatograms (EIC); substrate (S) signals are shown in green, cyclized core domains (C) in red, and linearized core domains (L) in blue. Percentages in boxes are the amount of product present as cyclized core domain as a percentage of total cyclic + linear products.


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1.4. Core Domain Length Requirement. Naturally-occurring cycloamanides in Amanita species contain 6 to 10 amino acids.18,20 To examine the allowed peptide lengths for cyclization by POPB in vitro, we prepared six precursor peptides in E. coli with core domains ranging from 6 to 16 residues. Core domains with less than 8 residues were prepared by removing amino acids from the wild type AMA1 sequence (some shorter peptides were also based on natural cycloamanides; see below). For longer sequences, Gly, Ala, and Val were added due to their small size and passive nature, and Ser was included in the 16mer sequence to avoid possible issues with water insolubility. Cyclization occurred for all tested substrates with longer core domains (9mer, 10mer, 12mer, and 16mer) (Figure 4). Longer sequences were less efficiently cyclized, but even the 16mer yielded 42% cyclic product with some unreacted substrate and some linear 16mer. Hexamer and heptamer core peptides were efficiently processed but only linear products were produced.

Figure 4. POPB products produced from substrates with varying core domain lengths. “% Cyclic” is the amount of product produced as cyclized core domain as a percentage of total cyclic + linear product. Shown are overlaid extracted ion chromatograms (EIC); substrate (S) signals are shown in green, cyclized core domains (C) in red, and linearized core domains (L) in blue.



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1.5. Synthesis of Naturally Occurring Cycloamanides. Amanita phalloides and A. bisporigera produce a number of homodetic monocyclic hexa- to decapeptides, of which six have been structurally characterized.18,20 The known mushroom genomes predict that these fungi produce more than 50 additional cycloamanides.19,20 We tested cyclization of POPB substrates containing the sequences of several cycloamanides produced by expression in E. coli, as well as sequences for the precursors of β-amanitin (i.e., α-amanitin in which Asp7 replaces Asn7), and two phallotoxins, phallacidin (PHA; core sequence AWLVDCP) and phalloidin (PHD; core sequence AWLATCP). As before, only linearized products from sequences shorter than eight residues were observed (i.e., CylA, CylB, PHA, and PHD) (Table 2, Figure S9). The N-terminal leader peptide of the substrate containing the phallacidin (PHA) sequence was hydrolyzed to yield the 25mer, but no further hydrolase or cyclase activity was observed. The inability of POPB to cyclize these shorter sequences was unexpected, since Amanita mushrooms make cyclic hexapeptides (CylA) and heptapeptides (CylB and phallotoxins). Possible explanations are that other steps such as hydroxylation or epimerization occur before, and are required for, cyclization by POPB, or that the enzyme from Galerina has more limited substrate versatility than POPB from Amanita species. All naturally occurring sequences with at least eight residues were cyclized with good yields including the β-amanitin sequence (Table 2, corresponding LS/MS traces in Figure S9). The decamer antamanide sequence was cyclized slowly, with less than 10% of the substrate being consumed after 4 hr incubation. Overall, these results show that POPB could be useful to produce at least some of the natural cycloamanides, which have immunosuppressive and other biological activities but are currently available in limited quantities only from mushroom extracts.31,32


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Table 2. Cyclization of naturally occurring cycloamanides. Corresponding LC/MS traces are shown in Figure S9. Substrate cycloamanide A (CylA) cycloamanide B (CylB) phallacidin (PHA) phalloidin (PHD) β-amanitin (BETA) cycloamanide C (CylC) cycloamanide D (CylD) antamanide (ANT)

Length 6 7 7 7 8 8 8 10

Sequence VFFAGP SFFFPIP AWLVDCP AWLATCP IWGIGCDP MLGFLVLP MLGFLPLP FFVPPAFFPP

% Cyclic

Versatility of Prolyl Oligopeptidase B in Peptide Macrocyclization

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