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Enzymatic crosslinking of side chains generates a modified peptide with four hairpin-like bicyclic repeats Hyunbin Lee, Youngseon Park, and Seokhee Kim Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00808 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 27, 2017
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Biochemistry
Enzymatic crosslinking of side chains generates a modified peptide with four hairpin-like bicyclic repeats Hyunbin Lee, Youngseon Park, and Seokhee Kim* Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea Supporting Information Placeholder ABSTRACT: Macrocyclization of peptides is often employed to generate novel structures and biological activities in biosynthesis of natural products and drug discovery. The enzymatic crosslinking of two side chains in a peptide via an ester or amide has a high potential for making topologically diverse cyclic peptides, but is found with only a single consensus sequence in the microviridin class of natural products. Here, we report that a peptide with a new sequence pattern can be enzymatically crosslinked to make a novel microviridin-like peptide, plesiocin, which contains four repeats of a distinct hairpin-like bicyclic structure and shows strong inhibition of proteases. A single ATP-grasp enzyme binds to a leader peptide, of which only 13 residues are required for binding, and performs eight esterification reactions on the core peptide. We also demonstrate that the combination of tandem mass spectrometry and an ester-specific reaction greatly facilitates the determination of connectivity. We suggest that the enzymatic crosslinking of peptide side chains can generate more diverse structures in nature or by engineering.
Natural products present many classes of complex molecules with diverse structures and biological activities, and have historically been a main source of drugs.1 Recent advances in genomics reveal that a new class of natural products with distinct biosynthetic pathways, ribosomally synthesized and post-translationally
modified peptides (RiPPs), makes up a large group of natural products.2-4 Enzymatic modifications of ribosomally synthesized peptides provide a simple but powerful route in generating molecules with diverse structure and function without an extensive biosynthetic gene cluster, and, therefore, show a great potential for expanding chemical diversity and developing new biologically active molecules. RiPPs often employ macrocyclization of precursor peptides for restricting conformational flexibility and increasing metabolic stability.2, 5 Members of the microviridin family of RiPPs contain distinct intramolecular ω-ester and ω-amide bonds that connect a carboxyl side chain of glutamate or aspartate with a hydroxyl side chain of threonine or serine, or with an amine side chain of lysine.6-12 Two homologous ligase enzymes carrying an ATPgrasp domain use the energy of ATP to make two esters and one amide, and generate a rigid tricyclic peptide with protease inhibition activity.13-16 This type of enzymatic crosslinking of peptide side chains provides a distinct way to generate new structures and biologically active molecules. Microviridins, however, have only one specific consensus sequence for the precursor peptide, TxKxPSDx(E/D)(D/E) (x means various amino acids), which significantly limits their structural diversity.12, 17 Here, we identify a new RiPP, plesiocin, that displays the microviridin-like modification in a precursor peptide of a different sequence pattern. We demonstrate that the enzymatic side chain
Figure 1. A new ribosomally synthesized peptide is modified by the ATP-grasp enzyme encoded from the same gene cluster. (A) The biosynthetic gene cluster for a new modified peptide found in Plesiocystis pacifica SIR-1. One additional gene (psnA2) for a homologous precursor peptide is distantly found. (B) Sequence alignment of the two precursor peptides, PsnA1 and PsnA2. They are composed of the leader peptide (red and blue) and the core peptide (violet). Blue residues represent the conserved double Gly motif. (C) Sequence alignment of the four core repeats of PsnA2. The core peptide has four “TTxxxxEE” motifs. (D and E) The PsnB enzyme (2 µM) and the PsnA2 peptide (40 µM, calc. m/z for [M+H]+ = 7485) were mixed in various conditions at 37 oC. After 40 minutes, the reaction mixtures were analyzed by MALDI (D) and HPLC (E). (F and G) The PsnB enzyme (0.4 µM) and the PsnA2 peptide (40 µM) were mixed for reaction, and the reaction solutions taken at designated time points were analyzed by MALDI (F) and HPLC (G).
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crosslinking via ester bonds naturally generates a novel peptide derivative with a distinct structure and protease inhibition activity, suggesting greater potential for diverse structures. One of the interesting candidate gene clusters for microviridinlike RiPPs was found in Plesiocystis pacifica, a marine myxobacterium. The gene cluster was predicted to contain three genes that encode a precursor peptide (psnA1), an ATP-grasp enzyme (psnB), and a membrane protein of unknown function (psnC) (Figure 1A).18 A homologous gene for the precursor peptide (psnA2) was also identified in a distant region of the same genome. Sequence analysis indicates that two precursor peptides may contain the N-terminal 24-25 residue long leader peptides for enzyme binding, and the C-terminal four repeats of 9-13 residue cores that may be modified by enzyme (Figure 1B). The core repeats of PsnA2 commonly contain two contiguous threonines and two contiguous glutamates (TTxxxxEE) for potential crosslinks, but three repeats also have additional aspartates (Figure 1C). Because the PsnA2 precursor peptide is shorter and has more regular consensus sequence for core repeats, we focused on the characterization of PsnA2. To determine whether the precursor peptide is modified by the ATP-grasp enzyme, we heterologously expressed and purified the PsnA2 precursor peptide and the PsnB enzyme in Escherichia coli. We incubated them in various conditions, analyzed the products with MALDI-TOF-MS and HPLC, and found that the reaction product lost 144 Da equivalent to eight water molecules and required the enzyme and ATP (Figure 1D and 1E). This result suggests that PsnA2 is indeed modified via PsnB-mediated condensation reactions. Incomplete reaction revealed various intermediates with mass losses equivalent to 1-7 water molecules, indicating that the peptide crosslinking reaction proceeds distributively with repetitive enzyme-substrate association and dissociation (Figure 1F and 1G). The complete reaction at 100 minute suggests that kcat of the reaction is higher than 1 min-1 at 37 oC, which is faster than the MdnC-mediated reaction in microviridin biosynthesis.19 We further dissected the modification of the precursor peptide and confirmed that the modified PsnA2 is indeed a novel microviridin-like RiPP with following observations: First, only the core region of the precursor peptide is modified. A tryptic fragment of the reaction product that contains the C-terminal core region (a*), named plesiocin, also lost 144 Da (Figure 2A and 2B). Second, crosslinking reactions occur only within each repeat and each of four core repeats contains two crosslinks. It has been reported that the amide bond between aspartate and proline (Asp-Pro) can be selectively cleaved by 70%(v/v) formic acid at ambient temperature.20 Four Asp-Pro bonds exist only between two neighboring core repeats in PsnA2 (Figure 2A, arrows). The formic acidmediated cleavage of plesiocin generated four doubly dehydrated core repeats (R1-R4), indicating that there is no inter-repeat crosslink and each core repeat contains two crosslinks (Figure 2C). Third, two chemical reactions provide evidences of ester bonds. The four modified core repeats (R1-R4) were subjected to either NaOH-mediated hydrolysis or NaOCH3-mediated methanolysis, and as expected, showed the addition of two water molecules or two methanol molecules, respectively (Figure 2D and S1). Finally, the crosslinking reaction occurs between two threonines and two glutamates. R1-R4, their hydrolysis products, and their methanolysis products were analyzed with MALDI-TOF-MS/MS. R1R4 showed the fragmentation patterns with two dehydrated threonines instead of two threonines, while the hydrolysis products present the normal fragmentation patterns for unmodified core peptides (Figure 2E, 2F, and S2), indicating that two threonines participate in the crosslinking reaction. The methanolysis products revealed two glutamate methyl esters instead of two glutamates in
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Figure 2. The modified PsnA2 peptide is a microviridin-like RiPP with eight ester crosslinks. Predicted and observed masses for all mass spectra are in Table S1. (A) Sequence of the PsnA2 precursor peptide. a and a* are tryptic fragments of the core peptide without or with modifications. R1-R4 are four fragments from the formic acid-mediated cleavage of a*. Arrows indicate the cleavage sites (Asp-Pro). (B) MALDI spectra of tryptic fragments of PsnA2 (black) or the modified PsnA2 (red). (C) A portion of the reaction solution, where plesiocin (a*) was cleaved with 70% formic acid, was taken at indicated time points and analyzed by MALDI. (D) R1 was treated with NaOH (500 mM) for hydrolysis or with NaOCH3 (10 mM) for methanolysis, and their reaction products were analyzed by MALDI. (E-G) R1 (E), NaOH-treated R1 (F), and NaOCH3treated R1 (G) were analyzed by MALDI-TOF-MS/MS. Several y-series (blue and dark blue) and b-series (orange and red) ions were observed in the spectra. Some y-series ions (dark blue) and b-series ions (red) indicate the distinct rearrangement or reactions shown in the top-right schemes. For example, two ester bonds in R1 are rearranged in the mass analyzer, resulting in two dehydrated threonines (E), and the methanolyzed R1 leaves two glutamate methyl esters (G).
the fragmentation patterns, suggesting that two glutamates are crosslinked (Figure 2G and S2). There are two possible topologies for each crosslinked core repeat: one with the hairpin-like structure and the other with the helix-like structure (Figure 3A). To determine the connectivity of two crosslinks in a repeat, we partially hydrolyzed each modified core repeat and the two partially hydrolyzed peptides carrying only one crosslink were analyzed with MALDI-TOF-MS/MS either directly or after methanolysis (Figure 3B). Results with four modified core repeats collectively suggest that plesiocin is a modified peptide with four hairpin-like bicyclic repeats (Figure 3C). In experiments with the first core repeat (R1), we found that the first threonine is connected to the second glutamate (Figure 3D and 3E) and the second threonine is crosslinked to the first glutamate (Figure 3F and 3G). We also confirmed that the rest of three core repeats (R2-R4) have the same connectivity (Figure S3). The leader peptide of PsnA2 has 25 residues, of which the last 12 residues are highly conserved between PsnA1 and PsnA2 (Fig-
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Biochemistry
Figure 3. Plesiocin has four repeats of a hairpin-like bicyclic peptide. (A) The core repeats with two ester crosslinks may have a hairpin-like or helix-like structure. The red line represents an ester crosslink. (B) Schematic of the workflow for determining the connectivity of the two ester crosslinks. Two partially hydrolyzed products are analyzed by the MS/MS either directly or after methanolysis. (C) A structural model for plesiocin, the leaderless modified PsnA2 peptide. Each core repeat is crosslinked with two ω-esters and forms a hairpin-like structure. Grey lines represent normal peptide bonds. (DG) R1 was analyzed as described in (B) by MALDI-TOF-MS/MS. Several y-series (blue and dark blue) and b-series (orange and red) ions were observed in the spectra. Some y-series ions (dark blue) and b-series ions (red) help determining which residues are crosslinked. Predicted and observed masses for mass spectra are in Table S1.
ure 1B). To determine an important region in the leader peptide for enzyme recognition, we tested several truncated variants for crosslinking reaction, and found that the deletion of less conserved ten residues at N-terminus (K3-R12) did not significantly deter the reaction (Figure S4). However, deletion of a more conserved region, D13LFIEDLGKV22, seriously hampered completing the reaction, indicating that this sequence is central for enzyme binding. Deletion of the glycine-rich sequence at C-terminus of the leader peptide generated a product that loses six water molecules, indicating that this flexible region may function as a linker. Collectively, 13 residues in the leader peptide are sufficient for the enzyme reaction. Many microviridins can inhibit various proteases.6-12, 15, 16 Because the sequences between threonines and glutamates are mostly hydrophobic in PsnA2, we tested plesiocin and a shorter fragment for inhibition of two proteases with hydrophobic P1 specificity, elastase and chymotrypsin. Although a single modified repeat (R1) showed much higher Ki values (380 and 63 nM, respectively), plesiocin could inhibit both proteases with low nanomolar Ki values (16 and 7.5 nM, respectively; Figure 4A and 4B). This result suggests that, although a single crosslinked core repeat
Figure 4. Plesiocin and its first modified core repeat (R1) can inhibit two proteases with hydrophobic P1 specificity. Cleavage of N-SuccinylAla-Ala-Ala-p-nitroanilide (1.5 mM) by elastase (0.5 nM; A), NSuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (77 µM) by chymotrypsin (0.25 nM; B), or Nα-Benzoyl-L-Arg 4-nitroanilide (300 µM) by trypsin (160 nM; C) was monitored with different concentrations of plesiocin (red) or R1 (black). The curves are fits to a hyperbolic equation. Error bars are averages ±1 SD (n = 3).
can inhibit proteases, multiple repeats greatly enhance the interaction with proteases. Of note, the inhibitory effect of plesiocin against elastase and chymotrypsin is comparable to or stronger than those of microviridins reported so far.12, 15, 16 By contrast, plesiocin does not inhibit trypsin, which has hydrophilic P1 specificity (Figure 4C). It is not clear how the leader peptide is cleaved and the modified core peptide is exported. However, some evidences suggest that the modified PsnA2 is exported after the leader cleavage by a type 1 secretion system (T1SS) as some RiPPs are in Gramnegative bacteria.21-23 First, the third gene product, PsnC, is ho mologous to HlyD, which is a component of T1SS and helps recognizing and transporting the secreted substrates (Figure S5).24 Second, the leader peptide of PsnA2 has a conserved double Gly motif (Figure 1B), which is recognized and cleaved by another component of T1SS, a peptidase-containing ATP-binding cassette transporter (PCAT).22, 25 Therefore, we suggest that a PCAT protein in P. pacifica may work with PsnC for the leader cleavage and the product secretion. To investigate the natural diversity of the plesiocin-like RiPPs, we used the BLAST search for genes that are homologous to psnA2 and are located nearby a gene for an ATP-grasp enzyme. We could identify nine new biosynthetic gene clusters in several species of proteobacteria, cyanobacteria and firmicutes (Figure S6). Their precursor peptides have up to fifteen repeats of the “TTxxxxEE” motif with diverse mid sequences. Of note, previous bioinformatic analysis showed that several precursor peptides of novel sequence patterns are associated with ATP-grasp enzymes in the same gene clusters, suggesting that more natural cyclic peptides with novel topologies may be generated by the side chain crosslinking via esters or amides.12, 18 In conclusion, we identified a novel microviridin-like RiPP, plesiocin, with strong protease inhibition activity. The ATP-grasp enzyme, PsnB, can generate four repeats of a hairpin-like bicyclic peptide by introducing two ester crosslinks in each repeat. This is the first example showing that a core sequence different from
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microviridins can be enzymatically crosslinked via intramolecular ω-esters. We propose that the enzymatic crosslinking of two side chains via an ester or amide has greater potential for expanding chemical diversity because it can make multiple crosslinks in a peptide and the different topological connectivity necessarily generates a different structure. By contrast, typical end-to-end or side-to-end cyclizations that are often found in other classes of RiPPs can generate only one ring topology.2 Furthermore, we suggest that the combination of the tandem mass spectrometry and an ester-specific reaction is a powerful approach to quickly determine the connectivity, and thus, promotes efficient identification and characterization of new members in this class of RiPPs.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental methods, supplementary figures and supplementary tables (PDF)
AUTHOR INFORMATION Corresponding Author *
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by Promising-Pioneering Researcher Program through Seoul National University (SNU). We thank Heejin Roh, Jungun Park, and Seokhee Lee for helpful discussions, and Yoonsoo Hwang, Hyunjin Cho, Sungjae Kim, Yurie T. Kim, and Gaeul Eom for chemicals and technical supports.
REFERENCES [1] Harvey, A. L., Edrada-Ebel, R., and Quinn, R. J. (2015) Nat. Rev. Drug Discov. 14, 111-129. [2] Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. A., Bugni, T. S., Bulaj, G., Camarero, J. A., Campopiano, D. J., Challis, G. L., Clardy, J., Cotter, P. D., Craik, D. J., Dawson, M., Dittmann, E., Donadio, S., Dorrestein, P. C., Entian, K.-D., Fischbach, M. A., Garavelli, J. S., Goransson, U., Gruber, C. W., Haft, D. H., Hemscheidt, T. K., Hertweck, C., Hill, C., Horswill, A. R., Jaspars, M., Kelly, W. L., Klinman, J. P., Kuipers, O. P., Link, A. J., Liu, W., Marahiel, M. A., Mitchell, D. A., Moll, G. N., Moore, B. S., Muller, R., Nair, S. K., Nes, I. F., Norris, G. E., Olivera, B. M., Onaka, H., Patchett, M. L., Piel, J., Reaney, M. J. T., Rebuffat, S., Ross, R. P., Sahl, H.-G., Schmidt, E. W., Selsted, M. E., Severinov, K., Shen, B., Sivonen, K., Smith, L., Stein, T., Sussmuth, R. D., Tagg, J. R., Tang, G.-L., Truman, A. W., Vederas, J. C., Walsh, C. T., Walton, J. D., Wenzel, S. C., Willey,
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J. M., and van der Donk, W. A. (2013) Nat. Prod. Rep. 30, 108160. [3] Ortega, Manuel A., and van der Donk, W. A. (2016) Cell Chem. Biol. 23, 31-44. [4] Cimermancic, P., Medema, Marnix H., Claesen, J., Kurita, K., Wieland Brown, Laura C., Mavrommatis, K., Pati, A., Godfrey, Paul A., Koehrsen, M., Clardy, J., Birren, Bruce W., Takano, E., Sali, A., Linington, Roger G., and Fischbach, Michael A. (2014) Cell 158, 412-421. [5] Truman, A. W. (2016) Beilstein J. Org. Chem. 12, 1250-1268. [6] Ishitsuka, M. O., Kusumi, T., Kakisawa, H., Kaya, K., and Watanabe, M. M. (1990) J. Am. Chem. Soc. 112, 8180-8182. [7] Okino, T., Matsuda, H., Murakami, M., and Yamaguchi, K. (1995) Tetrahedron 51, 10679-10686. [8] Shin, H. J., Murakami, M., Matsuda, H., and Yamaguchi, K. (1996) Tetrahedron 52, 8159-8168. [9] Murakami, M., Sun, Q., Ishida, K., Matsuda, H., Okino, T., and Yamaguchi, K. (1997) Phytochemistry 45, 1197-1202. [10] Fujii, K., Sivonen, K., Naganawa, E., and Harada, K.-i. (2000) Tetrahedron 56, 725-733. [11] Rohrlack, T., Christoffersen, K., Hansen, P. E., Zhang, W., Czarnecki, O., Henning, M., Fastner, J., Erhard, M., Neilan, B. A., and Kaebernick, M. (2003) J. Chem. Ecol. 29, 1757-1770. [12] Ahmed, M. N., Reyna-González, E., Schmid, B., Wiebach, V., Süssmuth, R. D., Dittmann, E., and Fewer, D. P. (2017) ACS Chem. Biol. 12, 1538-1546. [13] Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C., and Dittmann, E. (2008) Angew. Chem. Int. Ed. 47, 7756-7759. [14] Philmus, B., Christiansen, G., Yoshida, W. Y., and Hemscheidt, T. K. (2008) ChemBioChem 9, 3066-3073. [15] Weiz, A. R., Ishida, K., Quitterer, F., Meyer, S., Kehr, J.-C., Müller, K. M., Groll, M., Hertweck, C., and Dittmann, E. (2014) Angew. Chem. Int. Ed. 53, 3735-3738. [16] Reyna-González, E., Schmid, B., Petras, D., Süssmuth, R. D., and Dittmann, E. (2016) Angew. Chem. Int. Ed. 55, 9398-9401. [17] Philmus, B., Guerrette, J. P., and Hemscheidt, T. K. (2009) ACS Chem. Biol. 4, 429-434. [18] Iyer, L. M., Abhiman, S., Maxwell Burroughs, A., and Aravind, L. (2009) Mol. Biosyst. 5, 1636-1660. [19] Li, K., Condurso, H. L., Li, G., Ding, Y., and Bruner, S. D. (2016) Nat. Chem. Biol. 12, 973-979. [20] Piszkiewicz, D., Landon, M., and Smith, E. L. (1970) Biochem. Biophys. Res. Commun. 40, 1173-1178. [21] Havarstein, L. S., Diep, D. B., and Nes, I. F. (1995) Mol. Microbiol. 16, 229-240. [22] Duquesne, S., Destoumieux-Garzon, D., Peduzzi, J., and Rebuffat, S. (2007) Nat. Prod. Rep. 24, 708-734. [23] Furgerson Ihnken, L. A., Chatterjee, C., and van der Donk, W. A. (2008) Biochemistry 47, 7352-7363. [24] Balakrishnan, L., Hughes, C., and Koronakis, V. (2001) J. Mol. Biol. 313, 501-510. [25] Lin, D. Y.-w., Huang, S., and Chen, J. (2015) Nature 523, 425-430.
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Biochemistry
TOC Graphic
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5
A
B
PPSIR1_03888
psnA1
1 2 3 C4 Repeat 1 27 5 Repeat 2 40 6 Repeat 3 53 7 Repeat 4 63 8 Consensus 9 10
PPSIR1_03893
PPSIR1_03898
psnC
ATP-grasp Enzyme
Membrane Protein
D 39
PPSIR1_14310
200bp
Product (-144)
Consensus
Leader
Precursor
E Precursor
7485
Product
Core
F
Native
--P-TTLALGEED
62
Modified
7341
--P-TTLAIGEE
71
∆Enzyme
ACS Paragon Plus∆Enzyme Environment 20.25min
Native Modified
∆ATP 7300
∆ATP 7400 m/z
7500
8
10 12 14 Retention Time (min)
G
(x18Da) -7 -5 -3 -1 Product(-8) -6 -4 -2 Precursor Native
--PITTLAIGEEDPD 52
P TTLAhGEE
36
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PsnA1 37 PATTLALGEEEP-TTLSLQAEE--PTTLSLQAEEPTTLSLSAED 77 PsnA2 29 PYTTLAIGEEDPITTLAIGEEDPDPTTLALGEEDPTTLAIGEE 71 P TTLAhGEE+P TTL+h E+ PTTL+L E+PTTL+h. E Consensus
psnA2
psnB
GGPYTTLAIGEED
1 MS-NDKKNNELKELFIEDLGQVTGG-CRRPHPTPVGEG MSKNENNKKQLRDLFIEDLGKVTGG----------KGG MS N++++++L++LFIEDLG+VTGG G
PsnA1
Biochemistry PsnA2 1
Precursor Peptides
Precursor
1min
1min
4.5min
4.5min 20.25min
100min 7300
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Native
100min 7400 m/z
7500
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10 12 14 Retention Time (min)
a* = not modified APage 7 of 10 Biochemistry a* = fully modified (plesiocin)
= 2 ester linkages
GSKNENNKKQLRDLFIEDLGKVTGGK GGPYTTLAIGEED PITTLAIGEED PD PTTLALGEED PTTLAIGEE R1 R2 R3 R4
B1
C
R3+R4+PD R1+R2
D
a* a 2 R3+R4 R1+R2+PD R1+H a* (plesiocin) 2hr 3 R1+2H O+H 144 4 12hr R1+2CH OH+H R1 5 24hr +NaOH +K Precursor 6 R3 R2 R1 + Na Modified R4 48hr +NaOCH 7 4000 5000 1000 2000 3000 4000 5000 1250 1300 1350 1400 8 m/z m/z m/z H H E9 O O Mass analyzer y8 y7 y6 y5 y4 y3 y2 y1 y12y11y10 y9 10 O Thr O Thr Glu Glu y11 11 GGPY T T L A I G E E D b112 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b11 y9 b12 y12 [M+H] b5 b6 y6 b7 b8 y8 13R1 14 O OH F y12 y11y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 OH OH 15 O Thr Glu Glu H O Thr O G16 G P Y T T L A I G E E D b8 b9 b1b2 17b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b7 y8 [M+H] y6 y7 b11 b12 y11 b10 y5 b6 R1 + NaOH b5 18 G19 O OCH OH OCH y12 y11y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 20 O Thr Glu CH OHThr O Glu E D b8 G21 G PY T T L A I GE b9 b1 b2 22b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b6 y6 b7 y7 Environment [M+H] b11 ACS Paragon Plus b10 b12 23 b5 R1 + NaOCH 24 0 200 400 600 800 1000 1200 1400 m/z 25 +
+
2
+
3
+
+
3
(-H2O)
(-H2O)
+
-
2
+
-
3
3
(Methyl)
(Methyl)
3
+
3
L
O
T
O O
COOH
OH
OH
LA O
DP
COOH
TT
O
TTxxxxEE
G
E
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C
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AL
B
EE
A
COOCH3 COOH
DPDP
O
O
EE
O
AI
G
O
OH OH
COOH COOH
OH
OH OH
COOH COOCH3
OH
O
O
TT
COOH COOH
O
2
T
Partial Hydrolysis 1 TTxxxxEE T E Rn (n=1,2,3,4) TTxxxxEE Hairpin-like Structure TTxxxxEE EE 2 -C Methanolysis Methanolysis MS/MS MS/MS Rn-b OO Rn-a H 3 MS/MS Analysis MS/MS Analysis Analysis Analysis EE NH -GGPY TT 4 O O 5 TTxxxxEE EE O Helix-like Structure DPI T 6 O TTxxxxEE TTxxxxEE TTxxxxEE TTxxxxEE T E G D F L 7 L AIG 8 Plesiocin 9 D E y12 y11y10 y9 y8 y7y6 y5 y4 y3 y2 y1 10 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 y11 GGPYT TLA I GEE D b8 11G G P Y T T L A I G E E D b9 y7 b1 b2b3b4 b5 b6b7 b8b9 b10 b11 b12 b5 b6 b7 b8 b9 b10b11 b12 b1 b2 b3 b4 12 y7 b12 b8 [M+H] y5 b6 y6 b7 y8 b9 b10 y9 13 b5 b6 y6 b7 b10 b11 b12 y11 [M+H] R1-a R1-a Methanolysis b5 14 F15 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 G y12 y11y10 y9 y8y7y6 y5 y4 y3 y2 y1 G G P Y T T L A I G E E D b8 GGPY TT LA IGE ED 16 y11 b9 b1b2 b3b4 b5 b6 b7 b8b9 b10 b11 b12 y7 b6 b7 b8 b9 b10 b11 b12 b1b2 b3 b4 b5 b11 17 y9 b8 b12 y6 b7 y7 y8 y5 b6 y6 b7 b11 ACS Paragon Plus Environment b10 18 b12 y11 [M+H] [M+H] b5 b6 R1-b Methanolysis R1-b b5 19 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 m/z m/z 20 (Methyl)
(-H2O)
+
+
(-H2O)
(Methyl)
+
+
R1 KI = 380 nM
50
BBiochemistry C Chymotrypsin R1 KI = 63 nM
100 50
Trypsin
100
% activity
% activity
100
% activity
APage Elastase 9 of 10
50
plesiocin
1 No Inhibition Plus Environment plesiocin plesiocinACS Paragon 2 0 K =16 nM 0 K =7.5 nM 0 1 10 3 1 10 100 1000 10000 1 10 100 1000 Inhibitor (nM) Inhibitor (nM) Inhibitor (μM) 4 I
I
100
H
r
PsnB ATP
T
OO
O
H
O
O
O
T TT T ACS E T Paragon Plus Environment E
O
PsnA2
Plesiocin
T T EE
O
der
Lea
O
O -C
O O
O
E E
EE
O
OO
T T
EE
T
-C
E E
O
T
EE
Page 10 of 10 O TT
O
E
Le ad e
1 2 3 4 5 6 7
psnA2 Biochemistry E
O
psnB
TT
