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Screening, large-scale production and structure-based classification of cystine-dense peptides

Abstract

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria. Many showed extreme resistance to temperature, proteolysis and/or reduction, and all displayed inhibitory activity against at least 1 of 20 ion channels tested, thus confirming their biological functionality. Crystal structures of 12 confirmed proper cystine topology and the utility of crystallography to study these molecules but also highlighted the need for rational classification. Previous categorization attempts have focused on limited subsets featuring distinct motifs. Here we present a global definition, classification and analysis of >700 structures of cystine-dense peptides, providing a unifying framework for these molecules.

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Fig. 1: CDPs versus GFCKs.
Fig. 2: CDP biochemical and crystallographic analyses.
Fig. 3: Structure-based CDP-classification scheme.
Fig. 4: CDP sequence–structure relationships.
Fig. 5: Structure–sequence clustering of select subgroups of knotted CDPs.

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Acknowledgements

We thank J. Carter for substantial logistical support, and J. Simon, A. Mhyre and N. Nairn for helpful discussions. The project was supported by NIH grants R01CA135491-07 (J.M.O.) and T32-H600035 (C.D.B.), Project Violet, the Wissner-Slivka Foundation, the Kismet Foundation, the Sarah M. Hughes Foundation, Strong4Sam, Yahn Bernier and Beth McCaw, Len and Norma Klorfine, Anne Croco and Pocket Full of Hope.

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Contributions

C.E.C., M.M.G., C.M., M.-Y.B., D.M., J.M.O. and R.K.S. designed experiments, analyzed data, administered the project and wrote the manuscript. C.E.C., C.M., A.D.B., W.A.J., W.d.v.d.S. and S.M.T. developed the CDP production and analysis pipeline. M.M.G., P.B.R. and R.K.S. performed crystallographic structure determinations and analyses. C.E.C., M.M.G., M.-Y.B., D.M. and S.E.B. performed phylogenetic and taxonomic analyses. C.E.C., A.D.B., W.A.J., M.C., S.E.B., W.d.v.d.S., S.M.T., A.W. and M.K.C. produced and analyzed CDPs. C.E.C., M.M.G., K.P., C.D.B. and R.K.S. designed and developed SuperTEV.

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Correspondence to James M. Olson or Roland K. Strong.

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J.M.O. is a founder and shareholder of Blaze Bioscience, Inc.

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Integrated supplementary information

Supplementary Figure 1 CDP-production workflow.

An overview of the CDP production pipeline is presented, showing the sequential steps in the process. Step 1 is large-scale suspension culture of transduced HEK293F cells. Step 2 is purification, as depicted by PAGE analysis of an intact Scn-CDP fusion protein immediately following IMAC elution (non-reducing PAGE shows a single, highly-pure fusion protein). Step 3 is preparative cleavage with TEV protease (non-reducing SDS-PAGE shows near complete liberation of the CDP from the fusion construct). Step 4 is preparative-scale separation of the CDP from the Scn fusion partner using RPC. Step 5 is bulk lyophilization of the retentate. Step 6 is final analysis by comparative reduced/non-reduced PAGE and RPC. Note the aberrant mobility typical of purified CDPs in the comparative reduced/non-reduced PAGE analysis (inset, lower right, Step 6), where typical proteins with intrachain cystines migrate faster under non-reducing loading conditions than reducing ones.

Supplementary Figure 2 SuperTEV design, production and characterization.

a, A cartoon representation of the structure of TEV protease (1LVB; Phan, J. et al., J Biol Chem. 277, 50564–50572, 2002) is shown, with the positions of cysteine residues colored yellow on the ribbon, the position of a modified valine residue colored magenta, and asparagine residues at potential N-glycosylation sites shown as orange spheres. A peptide substrate is shown in a licorice-stick representation, colored by atom type. Cysteine residues were substituted as indicated to stabilize the protein, eliminate the need for reducing agents in storage/cleavage buffers, and permit expression through eukaryotic secretion pathways. V219 was replaced to stabilize the protein, based on analyses with Rosetta (Leaver-Fay, A. et al., Methods Enzymol. 487, 545–574, 2011). In order to prevent deleterious cryptic N-glycosylation, the indicated mutations were made at N23, to prevent a folding-incompatible N-glycan, and T173, to prevent steric occlusion of the active site by a non-native glycan on N171. Two other potential N-glycan sites (N68, N52) were not modified, as N-glycans at these positions would not be predicted to affect folding or activity. Combinations and variations of these mutations have been proposed before (Blommel, P.G. et al., Protein Expr Purif. 55, 53–68, 2007; Cabrita, L.D. et al., Protein Sci. 16, 2360–2367, 2007; Kapust, R.B. et al., Protein Eng. 14, 993–1000, 2001; Cesaratto, F. et al., J Biotechnol. 212, 159–166, 2015), though not combined into a single construct. Unlike native TEV, SuperTEV, produced in bacterial or mammalian expression platforms, was monodisperse by SEC, stable in storage, and more thermostable in solution (TmTEV = 52° C; TmSuperTEV = 56° C, as determined by CD). When produced in mammalian cells, SuperTEV showed a molecular weight increase consistent with glycosylation at N52 and N68, can be readily prepared free of contaminating endotoxins, and can be functionally co-expressed in the mammalian secretion pathway. b, TEV and SuperTEV show identical activities on a Scn-TEV site-target protein (CD86 ectodomain) fusion construct by PAGE analysis over a time course of 4 h (uncleaved fusion protein: purple triangle; cleaved Scn: red triangle; cleaved partner protein: blue triangle; time points at 0, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, 180, and 240 m). c, At top are shown schematic representations of the Daedalus Lentivirus SuperTEV and blue fluorescent protein fusion constructs, incorporating Igκ leader peptide (white box), linker (gray lines), Scn fusion partner (red circle), TEV scission site (green diamond), hexa-histidine purification tag (blue diamond), SuperTEV (green oval), and blue fluorescent protein (BFP) sequences (Subach, O.M. et al., Chem Biol. 15, 1116–1124, 2008). The Scn fusion is designed to optimize expression of SuperTEV in the Daedalus system, and the BFP fusion was engineered to confirm activity of co-expressed SuperTEV in mammalian culture. When both viruses are co-transduced into HEK293F cells, the culture supernatant contains efficiently-cleaved BFP, as shown in the fluorescing samples at the bottom of the frame. Well 1 shows media from untransduced cells; well 2: IMAC flow-through of media from cells transduced with Scn-BFP; well 3: IMAC eluate of media from cells transduced with Scn-BFP; well 4: IMAC flow-through of media from cells co-transduced with Scn-BFP and Scn-SuperTEV fusion proteins; well 5: IMAC eluate of media from cells co-transduced with Scn-BFP and Scn-SuperTEV.

Supplementary Figure 3 Comparisons of CDP structures.

CDP crystal structures determined as part of this work (target number indicated) are shown superimposed on the closest related structure available (PDB accession code indicated) in a backbone representation, colored by atom type, with explicit disulfide bonds. Carbon atoms from the determined crystal structures are colored gray, and from the related PDB structure, green. All molecules in the crystallographic AU, or the sheaf of top NMR solutions available in the deposited structure, were independently aligned and are shown. Red arrows highlight some of the cystines with conformations that significantly deviated between NMR and crystal structures, eg., an inversion of handedness.

Supplementary Figure 4 Ion-channel inhibitory activity of selected CDPs.

The effect of 37 selected CDPs on 20 ion channels at two dosages (0.2 or 20 μM) is shown with triangular gnomons, with grayscale intensity indicating the degree of the effect. Percent inhibition is defined as: (1-(ICDP /Icontrol)) x 100%. Individual assays were run in duplicate, and only effects greater than 3σ are shown. Though these results are more extensive than previously reported for many of these molecules, they are largely consistent with prior reports of ion channel activity for specific examples. CDPs #9 and #11 were active on a subset of potassium channels, consistent with previous reports (Laraba-Djebari, F. et al., J Biol Chem. 269, 32835–32843, 1994; Kozminsky-Atias, A. et al., FEBS Lett. 581, 2478–2484, 2007) and #9 is related to Kaliotoxin-1, a known Kv channel inhibitor, though #11 shows additional activity against hERG (Kv11.1) and Nav1.7 channels (not previously tested). CDP #14 had been previously reported to inhibit hERG channels (Restano-Cassulini, R. et al., Neurochem Res. 33, 1525–1533, 2008), but showed activity against additional potassium channels. CDP #21, aka Hadrucalcin, and CDP #55, aka Opicalcin-2, had been reported to inhibit calcium channels (ryanodine receptors) (Schwartz, E.F. et al., Br J Pharmacol. 157, 392–403, 2009; Xiao, L. et al., J Gen Physiol. 147, 375–394, 2016), but also showed acitivity against additional potassium channels or serotonin receptors (eg., 5-HT3a). CDP #28, CTX, was a known inhibitor of chloride channels (DeBin, J.A. et al., Am J Physiol. 264, C361-369, 1993), but also showed activity against the α4β2 nicotinic receptor, and the hERG and Kv1.2 potassium channels. CDP #46 had been previously reported to inhibit Kv1.3 (Mao, X. et al., Biochem Biophys Res Commun. 360, 728–734, 2007), consistent with these results. CDP #47 had been reported to inhibit small conductance, calcium-activated K channels (Romi-Lebrun, R. et al., Eur J Biochem. 245, 457–464, 1997), but not Kv channels as observed here. CDP #48 had previously been demonstrated to bind to hERG in the patent literature (US7326772 B2), but this confirmed hERG inhibition. CDPs #49 and #53 have been reported to inhibit Kv1.3 (Romi-Lebrun, R. et al., Biochemistry. 36, 13473–13482, 1997; Abdel-Mottaleb, Y. et al., Toxicon. 51, 1424–1430, 2008), and showed strong Kv1.3 inhibition in these results. CDP #62 and #63 showed activity on various subsets of potassium channels (D'Suze, G. et al., Arch Biochem Biophys. 430, 256–263, 2004; Legros, C. et al., FEBS Lett. 390, 81–84, 1996), #67 on voltage-sensitive calcium channels (Lampe, R. A. et al., Mol Pharmacol. 44, 451–460, 1993), and #86 showed activity on tetrodotoxin-sensitive sodium channels (eg., Nav1.7) (Peng, K. et al., J Biol Chem. 277, 47564–47571, 2002).

Supplementary Figure 5 Taxonomic distribution of CDPs and knotted CDPs in the PDB.

The distribution of CDPs (left column) and knotted CDPs (right column) by phylum (top row), class (middle row), and order (bottom row) identified in the PDB is shown as pie charts. Taxonomic division and percentage of total is labeled.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Note

Life Sciences Reporting Summary

Supplementary Table 1

Classification of experimentally-determined CDP structures in the PDB

Supplementary Table 2

Targeted CDP sequences, expression outcomes, properties, and biochemical characteristics

Supplementary Table 3

CDPs expressed using the high-throughput platform

Supplementary Table 4

Crystallization conditions and crystallographic validation statistics for CDP crystal structures

Supplementary Dataset 1

Properties of CDPs

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Correnti, C.E., Gewe, M.M., Mehlin, C. et al. Screening, large-scale production and structure-based classification of cystine-dense peptides. Nat Struct Mol Biol 25, 270–278 (2018). https://doi.org/10.1038/s41594-018-0033-9

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