Topological Regulation of the Bioactive Conformation of a Disulfide-Rich Peptide, Heat-Stable Enterotoxin

molecules Article Topological Regulation of the Bioactive Conformation of a Disulfide-Rich Peptide, Heat-Stable Enterotoxin Shigeru Shimamoto * , Mayu Fukutsuji, Toi Osumi, Masaya Goto, Hiroshi Toyoda and Yuji Hidaka * Faculty of Science and Engineering, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan; [email protected] (M.F.); [email protected] (T.O.); [email protected] (M.G.); [email protected] (H.T.) * Correspondence: [email protected] (S.S.); [email protected] (Y.H.); Tel.: +81-6-6721-2332 (S.S. & Y.H.) Academic Editor: Michio Iwaoka Received: 24 September 2020; Accepted: 16 October 2020; Published: 21 October 2020 Abstract: Heat-stable enterotoxin (STa) produced by enterotoxigenic E. coli causes acute diarrhea and also can be used as a specific probe for colorectal cancer cells. STa contains three intra-molecular disulfide bonds (C1–C4, C2–C5, and C3–C6 connectivity). The chemical synthesis of STa provided not only the native type of STa but also a topological isomer that had the native disulfide pairings. Interestingly, the activity of the topological isomer was approximately 1/10–1/2 that of the native STa. To further investigate the bioactive conformation of this molecule and the regulation of disulfide-coupled folding during its chemical syntheses, we examined the folding mechanism of STa that occurs during its chemical synthesis. The folding intermediate of STa with two disulfide bonds (C1–C4 and C3–C6) and two Cys(Acm) residues, the precursor peptide, was treated with iodine to produce a third disulfide bond under several conditions. The topological isomer was predominantly produced under all conditions tested, along with trace amounts of the native type of STa. In addition, NMR measurements indicated that the topological isomer has a left-handed spiral structure similar to that of the precursor peptide, while the native type of STa had a right-handed spiral structure. These results indicate that the order of the regioselective formation of disulfide bonds is important for the regulation of the final conformation of disulfide-rich peptides in chemical synthesis. Keywords: disulfide; enterotoxin; folding; guanylyl cyclase; topological 1. Introduction Disulfide bond formation is a post-translational modification that plays an important role in the stabilization of the native conformation of numerous peptides and proteins [1,2]. The correct disulfide pairings are typically required for the expression of biological activity. Disulfide-containing peptides and proteins are ideal models for studies of protein folding, since their folding intermediates can be observed, trapped, and separated by HPLC during the refolding reaction [3,4]. In addition, a chemical procedure for the regioselective formation of disulfide bonds make it possible to regulate and analyze the structures of the folding intermediates that frequently contain mis-bridged (non-native) or native disulfide bonds [5]. Heat-stable enterotoxin (STa) produced by enterotoxigenic E. coli is known as an exogenous ligand of the intestinal membrane receptor guanylyl cyclase-C (GC-C) and stimulates the secretion of chloride ions via the activation of cystic fibrosis transmembrane conductance regulator as well as the endogenous ligands, uroguanylin and guanylin, of the same receptor, GC-C [6,7]. These peptide hormones contain two disulfide bonds at positions similar to those for STa, which is crucial for biological activity, and they have been reported to be representative peptides that can be chemically Molecules 2020, 25, 4798; doi:10.3390/molecules25204798 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 11 Molecules 2020, 25, 4798 2 of 11 hormones contain two disulfide bonds at positions similar to those for STa, which is crucial for biological activity, and they have been reported to be representative peptides that can be chemically preparedprepared by by disulfide disulfide coupled coupled folding folding [ 8[8,9].,9]. ST STa ais is further further divided divided into into ST STh hand and ST STppderived derived from from a a humanhuman and and a a porcine porcine strain strain of of enterotoxigenic enterotoxigenicE. E. coli coli,, respectively respectively [ 10[10,11],,11], and and the the toxic toxic core core region region consistsconsists ofof 13 13 amino amino acid acid residues residues including including six six Cys Cys residues residues that that form form intra-molecular intra-molecular disulfide disulfide bonds,bonds, asas shownshown in Figure Figure 1 [[8,9,12].8,9,12]. The The reduced reduced form form of ST ofa STspontaneouslya spontaneously folds folds into only into the only native the nativeconformation conformation with the with correct the correct pairings pairings of their of disulfide their disulfide bonds bonds(C1–C4, (C1–C4, C2–C5, C2–C5, and C3–C6) and C3–C6) by air- byoxidation air-oxidation without without the need the for need any for mercapto-reagents, any mercapto-reagents, such as such glutathione, as glutathione, indicating indicating that the that amino the aminoacid sequence acid sequence of STa ofcarries STa carries sufficient suffi informationcient information to allow to allowthe molecule the molecule to fold to only fold into only the into native the nativeconformation. conformation. Thus, Thus,STa possesses STa possesses a rigid a conformation rigid conformation for a small for a peptide, small peptide, and therefore, and therefore, it would it wouldbe a good be a model good model for studies for studies of disulfi of disulfide-coupledde-coupled peptide peptide and protein and protein folding. folding. (a) (b) Figure 1. (a) Amino acid sequences and disulfide linkages of STh and STp produced by a human Figure 1. (a) Amino acid sequences and disulfide linkages of STh and STp produced by a human and and a porcine strain of enterotoxigenic E. coli, respectively. (b) The schematic drawing of STh(6–18). a porcine strain of enterotoxigenic E. coli, respectively. (b) The schematic drawing of STh(6–18). The The Cys6, Cys7, Cys10, Cys11, Cys15, and Cys18 residues were represented by C1, C2, C3, C4, C5, and Cys6, Cys7, Cys10, Cys11, Cys15, and Cys18 residues were represented by C1, C2, C3, C4, C5, and C6, respectively. C6, respectively. In our previous studies involving the chemical synthesis of STa peptides, STa was synthesized as In our previous studies involving the chemical synthesis of STa peptides, STa was synthesized as not only the native type of STa but also as the topological isomer with native disulfide pairings formed not only the native type of STa but also as the topological isomer with native disulfide pairings formed by the stepwise formation of disulfide bonds, although only the native type of STa was produced by theby simultaneousthe stepwise formation ofof threedisulfide disulfide bond bondss, although using only air-oxidation. the native Interestingly, type of STa was the toxicproduced activity by the simultaneous formation of three disulfide bonds using air-oxidation. Interestingly, the toxic of the topological isomer was still 1/10–1/2 that of the native STa [8]. The topological isomer was predominantlyactivity of the producedtopological during isomer the was regioselective still 1/10–1/2 formation that of the of native the disulfide STa [8]. bonds, The topological regardless isomer of the orderwas predominantly of the formation produced of the disulfide during bridges.the regioselecti It is particularlyve formation noteworthy of the disulfide that only bonds, the topological regardless of the order of the formation of the disulfide bridges. It is particularly noteworthy that only the isomer was obtained when the C2–C5 disulfide bond was formed by I2-oxidation at the final step [8,9]. Thetopological topological isomer isomer was is obtained stable in when the usual the C2–C5 buffers disulfide or organic bond solvents was formed without by mercapto-reagents, I2-oxidation at the final step [8,9]. The topological isomer is stable in the usual buffers or organic solvents without although the isomer is immediately converted into the native conformation of STa in the presence of thiolmercapto-reagents, reagents such as although mercaptoethanol. the isomer Theseis immediately observations converted suggest into that the the native topological conformation isomer of is ST a a kineticallyin the presence trapped of product thiol reagents that is producedsuch as merc duringaptoethanol. the chemical These synthesis, observations and that suggest regioselective that the disulfidetopological formation isomer providesis a kinetically a product trapped that product has a diff thaterent is conformationproduced during from the the chemical native peptide synthesis, in spiteand ofthat the regioselective fact that it contains disulfide the formation same disulfide provides bonds. a product that has a different conformation from the native peptide in spite of the fact that it contains the same disulfide bonds. Thus, the regioselective formation of the disulfide bonds of the STa peptide produces not only Thus, the regioselective formation of the disulfide bonds of the STa peptide produces not only the topological isomer but the native type of STa as well, thus providing an ideal model for studies ofthe peptide topological and protein isomer foldingbut the thatnative occur type during of STa chemicalas well, thus synthesis. providing Inaddition, an ideal model it should for bestudies noted of thatpeptide it might and notprotein be possible folding to that synthesize occur during the native

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