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 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 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 chemical type synthesis. of a target In peptide addition, or ait protein should thebe noted stepwise that formationit might not of disulfidebe possible bonds to synthesize because ofthe the native production type of ofa topologicaltarget peptide isomers or a protein [13,14]. the Therefore, stepwise toformation elucidate of the disulfide mechanism bonds responsible because forof the the producti folding ofon the of topologicaltopological isomerisomers and [13,14]. the native Therefore, form of to elucidate the mechanism responsible for the folding of the topological isomer and the native form of peptides that occur during the chemical synthesis, the precursor peptide of the topological isomer of STa waspeptides treated that with occur iodine during to form the thechemical third disulfidesynthesis, bond the precursor under several peptide sets of of the conditions. topological In addition,isomer of STa was treated with iodine to form the third disulfide bond under several sets of conditions. In

Molecules 2020, 25, x FOR PEER REVIEW 3 of 11 addition, NMR measurements of the precursor and the topological isomer were also carried out to obtainMolecules structural2020, 25, 4798 information on peptide and protein folding that occurs during the chemical3 of 11 procedures. NMR measurements of the precursor and the topological isomer were also carried out to obtain 2. Results structural information on peptide and protein folding that occurs during the chemical procedures.

2.1.2. Results Preparation of the Precursor Peptide with Two Disulfide Bonds and 2 × Cys(Acm) and the Topological Isomer 2.1. Preparation of the Precursor Peptide with Two Disulfide Bonds and 2 Cys(Acm) and the Topological IsomerWe previously reported that the topological isomer of STh(6–18)× was unexpectedly synthesized by stepwise regioselective disulfide formation, regardless of the order of the formation of three disulfideWe previously pairings [8]. reported Interestingly, that the topologicalonly the topological isomer of isomer STh(6–18) was was produced unexpectedly in the synthesizedcase of the byI2- oxidationstepwise regioselective of a precursor disulfide peptide that formation, contains regardless two Cys(Acm) of the orderresidues of theat the formation C2 and ofC5 three positions disulfide and pairingstwo disulfide [8]. Interestingly, bonds (C1–C4 only the and topological C3–C6 isomer connecti wasvity). produced Therefore, in the casethe of precursor the I2-oxidation peptide, of a precursor[Cys(Acm) peptide7,15,2SS]-ST thath(6–18), contains was two used Cys(Acm) to investigate residues the at folding the C2 andmechanism C5 positions of ST anda in twothe chemical disulfide 7,15 synthesisbonds (C1–C4 and andchemically C3–C6 connectivity).synthesized by Therefore, the Boc solid the precursor phase method peptide, (Figure [Cys(Acm) S1). ,2SS]-STh(6–18), wasAfter used todeblocking investigate the the protected folding peptide mechanism resins of STwitha in HF, the the chemical disulfide synthesis bonds were and chemicallyrandomly formedsynthesized by air-oxidation. by the Boc solid The phasetarget methodprecursor (Figure peptide S1). with two disulfide bonds at C1–C4 and C3–C6 and 2After × Cys(Acm) deblocking was the the protected major product peptide and resins was with purified HF, the by disulfide RP-HPLC, bonds as wereshown randomly in Figures formed S2A by air-oxidation. The target precursor peptide with two disulfide bonds at C1–C4 and C3–C6 and 2 and S3. The purified precursor peptide was further treated with iodine under the standard condition× usingCys(Acm) 50% MeOH was the to major provide product the topological and was purified isomer (Figure by RP-HPLC, S2C), as as previously shown in described Figures S2A [8]. and S3. The purifiedIn ordinary precursor conditions peptide of I2 was-oxidation, further only treated a single with iodinepeak was under observed the standard with the condition same relative using intensity50% MeOH of that to provide of the theprecursor topological peptide isomer on HPLC, (Figure as S2C), shown as previously in Figure 2a–c. described The retention [8]. time of this peakIn ordinary was obviousl conditionsy different of I2-oxidation, from that onlyof native a single form peak of ST wash(6–18). observed To estimate with the the same secondary relative structureintensity of thatthe topological of the precursor isomer peptide of STh(6–18) on HPLC, in this as peak shown fraction, in Figure circular2a–c. dichroism The retention (CD) timespectra of werethis peak obtained was obviouslyand compared different with from that of that the of nati nativeve form form (Figure of STh (6–18).S5). The To spectra estimate of the the peptide secondary in Figurestructure 2b,c of theshowed topological a smaller isomer ellipiticity of STh(6–18) at around in this 200 peak nm fraction, compared circular to that dichroism of the native (CD) spectra form, weresuggesting obtained that andthe ST comparedh(6–18) peptide with that obtained of the by native the present form (Figure methods S5). is Thethe topological spectra of theisomer peptide that hasin Figure a different2b,c showed structure a smallerfrom that ellipiticity of the native at around form. Thus, 200 nm the compared topological to isomer that of is the only native produced form, duringsuggesting chemical that the synthesis STh(6–18) by peptide the regioselective obtained by method the present with methods no traces is the of topologicalthe native isomertype ST thata being has observed.a different structure from that of the native form. Thus, the topological isomer is only produced during chemical synthesis by the regioselective method with no traces of the native type STa being observed.

Figure 2. RP-HPLC profiles of (a) Acm -precursor peptide and the reaction solutions after I -oxidation Figure 2. RP-HPLC profiles of (a) Acm2-precursor peptide and the reaction solutions after I2-oxidation in ((b)) 50%50% AcOH,AcOH, ( c(c)) 50% 50% MeOH MeOH/0.1/0.1 M M HCl, HCl, (d )(d 50%) 50%i-PrOH i-PrOH/0.1/0.1 M HCl,M HCl, and and (e) 80%(e) 80%i-PrOH i-PrOH/0.1/0.1 M HCl M with a linear gradient from 5 to 50% CH CN in 45 min (l.0%/min). The asterisks (*) are the position of HCl with a linear gradient from 5 to 50% 3CH3CN in 45 min (l.0%/min). The asterisks (*) are the position the expected retention time of the native type ST (6–18). of the expected retention time of the native type hSTh(6–18).

2.2. Topological Selection for the Formation of the Third Disulfide Bond Using I2-Oxidation 2.2. Topological Selection for the Formation of the Third Disulfide Bond Using I2-Oxidation The regioselective formation of disulfide bonds in peptide syntheses usually use a combination of The regioselective formation of disulfide bonds in peptide syntheses usually use a combination air-oxidation in the first step and I2-oxidation in the second step to avoid disulfide exchange reactions. of air-oxidation in the first step and I2-oxidation in the second step to avoid disulfide exchange Air-oxidation is typically carried out in an aqueous buffer at a slightly alkaline pH. However, because reactions. Air-oxidation is typically carried out in an aqueous buffer at a slightly alkaline pH. I2 is soluble in certain organic solvents, such as methanol, the reaction can be carried out in an organic However, because I2 is soluble in certain organic solvents, such as methanol, the reaction can be solvent. The velocity and yield of disulfide bond formation are dramatically affected by the type of organic solvent [15]. Therefore, to investigate the disulfide-coupled folding of peptides and proteins Molecules 2020, 25, x FOR PEER REVIEW 4 of 11 carried out in an organic solvent. The velocity and yield of disulfide bond formation are dramatically affected by the type of organic solvent [15]. Therefore, to investigate the disulfide-coupled folding of peptides and proteins that occur during chemical synthesis, solvents (AcOH, methanol, i-PrOH, and tetrahydrofuran) were investigated for the topological selection of STa. Disulfide bond formation proceeded simultaneously with the release of the Acm groups by I2-oxidation. In all acidic conditions investigated for I2-oxidation, the topological isomer was predominantly produced with only trace amounts of the native type of STa, as shown in Figure 2. These results suggest that the formation of the topological isomer depends on the conformation of the precursor peptide, suggesting that the Moleculeslocal conformation2020, 25, 4798 is stabilized by hydrogen bonds and/or the bulkiness of the molecular4 of 11 conformation at the transitional states are important for the topological selection of STa folding to occur during chemical synthesis. Actually, these solvents provide lower permittivity for the chemical thatreaction occur and during induce chemical hydrogen synthesis, bond formation solvents (AcOH, under the methanol, acidic conditionsi-PrOH, and as used tetrahydrofuran) in this study. were investigatedThe ionization for the topologicalof the N-terminal selection amino of ST groupa. Disulfide and the bond carboxyl formation group proceeded at the C-terminus simultaneously or on withside thechains, release such of as the Glu8 Acm in groups STh(6–18), by I2 in-oxidation. precursor In peptide all acidic are conditions affected by investigated the pH of the for solvent I2-oxidation, being theused. topological Therefore, isomer to estimate was predominantly the pH dependence produced of with topological only trace selection amounts in of the the folding native type of ST ofa, ST wea, asfurther shown examined in Figure 2disulfide. These results formation suggest in that50% theMeOH formation at pH of 7 theand topological 9. The I2-oxidation isomer depends of the onAcm the2- conformationprecursor peptide of the precursorpredominantly peptide, yielded suggesting the topologi that thecal local isomer conformation as well isas stabilized in the case by hydrogenat pH 2, bondsalthough and the/or yields the bulkiness of the topological of the molecular isomer conformation were lower at at both the transitionalpH 7 and 9 statescompared are important to that at pH for the2 (Figure topological 3A). These selection results of STsuggesta folding that to local occur ionic during interactions chemical are synthesis. not significant Actually, for the these topological solvents provideselection lower of ST permittivitya folding during forthe chemical chemical synthesis. reaction and induce hydrogen bond formation under the acidicTemperature conditions as is used also the in this important study. factor that affects reaction velocity and the extent of disulfide bondThe formation ionization during of the chemical N-terminal synthesis. amino group The increased and the carboxyl reaction group temperature at the C-terminus increases or not on only side chains,molecular such oscillations as Glu8 in STof ha(6–18), molecule in precursor in solution peptide but also are the affected flexibility by the of pH the of structure the solvent of a being precursor used. Therefore,peptide. To to investigate estimate the the pH effects dependence of temperature of topological on I2-oxidation, selection reaction in the foldingtemperatures of STa ,were we further varied examinedfrom 30 to disulfide80 °C. As formation shown in inFigure 50% MeOH3B, the topological at pH 7 and isomer 9. The was I2-oxidation the predominant of the Acm product2-precursor by I2- peptideoxidation predominantly at all temperatures, yielded thealthough topological the isomeryield of as wellthe topological as in the case isomer at pH decreased 2, although in the a yieldstemperature-dependent of the topological isomermanner were and lowerside reactions, at both pH in 7cluding and 9 comparedthe methylation to that atof pHcarboxyl 2 (Figure groups,3A). Thesewere clearly results observed suggest that at temperatures local ionic interactions over 50 °C. are These not significant results suggest for the that topological the conformation selection ofof STthea foldingprecursor during peptide chemical is tightly synthesis. restricted by two disulfide bonds.

Figure 3. (a) RP-HPLC profiles of Acm2-precursor peptide and the reaction solutions after I2-oxidation in 50% MeOH at pH 2, pH 7, and pH 9 with a linear gradient from 10 to 50% CH3CN in 40 min (l.0%/min). (b) RP-HPLC profiles of Acm2-precursor peptide and the reaction solutions after I2-oxidation in 50% tetrahydrofuran (THF) at 25–80 ◦C with a linear gradient from 10 to 40% CH3CN in 60 min (0.5%/min) after 10 min of injection.

Temperature is also the important factor that affects reaction velocity and the extent of disulfide bond formation during chemical synthesis. The increased reaction temperature increases not only molecular oscillations of a molecule in solution but also the flexibility of the structure of a precursor peptide. To investigate the effects of temperature on I2-oxidation, reaction temperatures were varied Molecules 2020, 25, 4798 5 of 11

from 30 to 80 ◦C. As shown in Figure3B, the topological isomer was the predominant product by I2-oxidation at all temperatures, although the yield of the topological isomer decreased in a temperature-dependent manner and side reactions, including the methylation of carboxyl groups, were clearly observed at temperatures over 50 ◦C. These results suggest that the conformation of the precursor peptide is tightly restricted by two disulfide bonds.

2.3. The Solution Structures of the Topological Isomer and Acm2-Precursor Peptide Determined by NMR Spectroscopy To obtain structural information regarding folding, we carried out NMR measurements of the topological isomer and the Acm2-precursor peptide of STh(6–18). NMR measurements of the Acm2-precursor peptide were performed in a 50% CD3COOD/50% D2O or a 50% CD3COOH/10% D2O mixture at pH 3 to avoid methylation in the acidic methanol solvent that was used for I2-oxidation (Figure S4). The peptide conformations related to topological selection were not affected by the types of buffers or solvents, as described above. Correlation Spectroscopy (COSY) and Nuclear Overhauser Effect Spectroscopy (NOESY) spectra were acquired and used for proton assignment and to obtain distance constraints. Using the CNS program, 2000 structures were calculated, and 10 structures with the lowest total energy structures were selected, as shown in Figure S6. The overall average root mean square deviation (RMSD) values for the backbone heavy atoms and all heavy atoms, including side chains of topological isomer and the Acm2-precursor peptide, are summarized in Table S1. The RMSD value for the STa molecule was quite low, since the three intra-molecular disulfide bonds make the STa molecule much more rigid, and therefore, the structural calculation provided such a small RMSD value. The superposition of the structures shows that the calculated structures of the topological isomer clearly converged (Figure S6), suggesting that the structure of the topological isomer is rigidly maintained by virtue of its intra-molecular disulfide bonds as well as that of the native form. On the other hand, the Acm2-precursor peptide also showed a well-converged backbone structure, but its side chains, especially the Acm groups at C2 and C5, were relatively disordered, suggesting that the Acm groups might be flexible in this solution (Figure S7). This result was consistent with the observation that the HN of Acm groups could not be defined due to signal broadening. The structure of the native form of STa was determined by X-ray crystallography in a previous study [16]. Based on the reported structure, the backbone of the native form adopts a right-handed spiral when seen from the N- to the C-terminal (Figure4A). In contrast, the structure of the topological isomer of STh(6–18) adopted a left-handed spiral backbone structure that was dramatically different from that of the native form, as shown in Figure4B. The native form of ST a consists of three β-turn moieties of which Cys7(C2)–Cys10(C3) and Asn12–Cys15(C5) form the type I β-turns and Cys15(C5)–Cys18(C6) forms the type II β-turn [16]. However, the topological isomer possessed only one β-turn (type I) in the Asn12–Cys15(C5) region, as shown in Figure5. Importantly, the structure of this region, corresponding to the receptor binding site, of the topological isomer, was similar to that of the native type STa, indicating that the biologically active conformation is still maintained in the structure of the topological peptide. MoleculesMolecules 20202020,, 2525,, x 4798 FOR PEER REVIEW 66 of of 11 11 Molecules 2020, 25, x FOR PEER REVIEW 6 of 11

Figure 4. The schematic drawings (left and middle) of (a) the native form, (b) topological isomer, and FigureFigure 4. 4. TheThe schematic schematic drawings drawings (left (left andand middlemiddle) of) ( ofa) the (a) thenative native form, form, (b) topological (b) topological isomer, isomer, and (c) Acm2-precursor peptide of STh(6–18). Their backbone structures were illustrated by cartoon and (c) Acm2-precursor peptide of STh(6–18). Their backbone structures were illustrated by cartoon representations.(c) Acm2-precursor The peptideorange spheres of ST hrepresent(6–18). Their the Cbackboneα of Cys. structuresThe disulfide were linkages, illustrated C1–C4, by cartoonC2–C5, representations. The orange spheres represent the Cα of Cys. The disulfide linkages, C1–C4, C2–C5, andrepresentations. C3–C6, were The illustrated orange spheres by stick represent representation the Cα ofcolored Cys. The by disulfideblue, red, linkages,and green C1–C4,, respectively. C2–C5, and C3–C6, were illustrated by stick representation colored by blue, red, and green, respectively. Schematicand C3–C6, drawings were illustrated of each structureby stick representation(right) showed colored the right by orblue the, red left-handed, and green spiral, respectively. of their Schematic drawings of each structure (right) showed the right or the left-handed spiral of their backbone backboneSchematic when drawings seen fromof each the N-structure to the C-terminal.(right) showed the right or the left-handed spiral of their backbonewhen seen when from seen the N- from to thethe C-terminal.N- to the C-terminal.

FigureFigure 5. 5. TheThe receptor receptor binding binding site site (-Asn-Pro-Ala-) (-Asn-Pro-Ala-) of of ST STa awaswas shown shown in in (a (a) )the the native native form form and and ( (bb)) topologicalFiguretopological 5. The isomer isomer receptor illustrated illustrated binding by by site lightlight (-Asn-Pro-Ala-) green green andand lightlight oforange orange STa ,was, respectively. respectively. shown in ( ((cca)) )The Thethe superpositionnative superposition form and of of the( theb) receptortopologicalreceptor binding binding isomer site site illustrated of of the the native native by light form form green and and andthe the topologicallight topological orange, isomer. isomer.respectively. (c) The superposition of the receptor binding site of the native form and the topological isomer. The NMR measurements revealed that the Acm2-precursor peptide also showed that the molecule The NMR measurements revealed that the Acm2-precursor peptide also showed that the moleculehas aThe left-handed hasNMR a left-handedmeasurements spiral backbone spiral revealed structure,backbone that similarstructure, the toAcm the similar2 topological-precursor to the isomertopologicalpeptide (Figure also isomer 4showedC). These(Figure that results 4C).the Thesemoleculewere consistentresults has werea left-handed with consistent previously spiral with reported backbone previously results, structure, repo showingrted similar results, that to the showing the CD topological spectra that ofthe isomer the CD Acm spectra (Figure2-precursor of 4C).the Thesepeptide results were similarwere consistent to that of with the topological previously isomerreported of STresults,p(5–17) showing (Figure that S5) [the14]. CD The spectra Asn12–Ala14 of the Acm2-precursor peptide were similar to that of the topological isomer of STp(5–17) (Figure S5)[14]. Acmregion2-precursor in the Acm peptide2-precursor were moleculesimilar to formedthat of the a γ -turntopological structure. isomer The of topological STp(5–17) isomer(Figure containsS5)[14]. The Asn12–Ala14 region in the Acm2-precursor molecule formed a γ-turn structure. The topological Theonly Asn12–Ala14 three backbone region hydrogen in the bonds,Acm2-precursor although themolecule native formed form of a ST γa-turnpossesses structure. five hydrogenThe topological bonds isomer contains only three backbone hydrogen bonds, although the native form of STa possesses five hydrogenisomer contains bonds only in itsthree backbone backbone structure hydrog en(Table bonds, S2). although Two of the the native three form hydrogen of STa possessesbonds in fivethe hydrogen bonds in its backbone structure (Table S2). Two of the three hydrogen bonds in the

Molecules 2020, 25, 4798 7 of 11 in its backbone structure (Table S2). Two of the three hydrogen bonds in the topological isomer were also retained in the Acm2-precursor peptide (Table S2). These results suggest that the left-handed spiral backbone structures of the topological and the Acm2-precursor peptides are stabilized, not only by hydrogen bonds, but they are also significantly restricted by the two disulfide bonds.

3. Discussion

The regioselective formation of the disulfide-rich peptide STa produced the topological isomer and regulated the conformation of the peptide. To elucidate the folding mechanism of the topological isomer during the chemical synthesis, the disulfide bonds of STa peptides were formed by a stepwise method using a combination of air-oxidation and I2-oxidation. The Acm2-precursor peptide with two disulfide bonds was treated with iodine under several conditions. The recovery of the topological isomer was improved when the I2-oxidation was carried out under acidic conditions; however, the topological isomer was exclusively produced from the Acm2-precursor peptide regardless of the pH value (Table1 and Figure3A).

Table 1. Recoveries for the I2-oxidation of the Acm2-precursor peptide.

Organic Solvents pH Temperature Recovery (%) a 50% AcOH 3 25 82 50% MeOH/0.1 M HCl 2 25 74 50% MeOH 7 25 30 50% MeOH/0.1 M NH3 aq 9 25 28 50% i-PrOH/0.1 M HCl 2 25 63 80% i-PrOH/0.1 M HCl 2 25 62 50% THF/0.05% TFA 2 25 71 50% THF/0.05% TFA 2 30 69 50% THF/0.05% TFA 2 40 64 50% THF/0.05% TFA 2 50 54 50% THF/0.05% TFA 2 60 36 50% THF/0.05% TFA 2 70 25 50% THF/0.05% TFA 2 80 19 a 7,15 The Acm2-precursor peptide, [Cys(Acm) ,2SS]-STh(6–18), was treated with iodine under various conditions. The recoveries of the topological isomer were determined by HPLC analysis.

It is known that uroguanylin, which is structurally homologous to STa (Figure1A), with two disulfide linkages (C2–C5 and C3–C6 connectivity) exhibits conformational switching between the native form and the topological isomer under acidic conditions [17,18] but not at neutral pH. Uroguanylin is sufficiently flexible to undergo a conformational change, and the protonation of carboxylate groups appears to support this conformational exchange. On the other hand, the Acm2-precursor peptide with two disulfide linkages (C1–C4 and C3–C6 connectivity) was detected as a single conformer on HPLC (Figure S2B), which may contain sufficient information to allow the topological isomer to be formed irrespective of the pH. In addition, the synthetic yield of the topological isomer decreased in a temperature-dependent manner, but the topological isomer was still obtained predominantly (Figure3B). Moreover, only the topological isomer was obtained by I 2-oxidation in the all solvents used in these experiments, as shown in Figure2. Alcohols with low dielectric constants are widely used as an inducer of α-helical structures and stabilizer of β-turns in peptides due to the artificial enhancement in intra-molecular hydrogen bonds. Our results suggest that the dielectric constant of the solvent or the temperature used had no effect on the topological selection of STa folding, indicating that the conformation of the Acm2-precursor peptide is restricted by two disulfide linkages and/or two Acm groups. This observation is consistent with the results of CD measurements indicating that the spectra of the Acm2-precursor peptide were nearly the same when several buffers were used (Figure S5). Molecules 2020, 25, 4798 8 of 11

The structure of the native form of STa has been determined by X-ray crystallography [16]. However, sufficient structural information on the topological isomer is lacking. To obtain further insights into the folding of the topological isomer, we determined the structures of the topological isomer and Acm2-precursor peptide. As shown in Figure4, the topological isomer had a clearly different structure from that of the native form in spite of the fact the both structures contained the same disulfide bonds and possessed a left-handed spiral structure, as seen from the N-terminal. These observations suggest that the topological isomer is a kinetically trapped product that is produced during chemical synthesis, and that the regioselective disulfide formation provides a product with a different conformation from the native peptide in spite of the fact that both contain the same disulfide bonds. The native type of STa requires a right-handed spiral structure for the folding process. Based on the NMR structure of the Acm2-precursor molecule, the C- or N-terminal moieties need to pass through the inside of the Acm2-precursor molecule, the structure of which is formed by the backbone structure and intra-molecular disulfide bonds, in order for the dynamic inter-conversion between the left- and right-handed spiral structure to occur. However, it is possible that the side chains of the Asn12 and Thr16 residues that are buried in the cavity in the spiral structure could interrupt the inter-conversion. The Acm group of Cys also interrupts the inter-conversion by virtue of its bulkiness, the size of which is comparable to that of the side chain of an Arg residue, and therefore, steric hindrance could occur between the Acm groups and other side chains, such as Asn12 and Thr16. As reported previously, the native type of STa is produced as a minor product regardless of the order of the regioselective disulfide bond formation [8]. It has been reported that the precursor peptide, STp(5–17) with two disulfide bonds at C2–C5 and C3–C6, showed two conformers on HPLC, and one conformer and another conformer yielded the topological isomer and the native type STp peptide, respectively [9]. Combined with this observation and the results in these experiments, it appears that the precursor peptides are kinetically trapped during the chemical synthesis and regulate subsequent disulfide formation at the last step in its folding. In addition, our results also raise an alarm that the synthesis of a disulfide-rich peptide by the regioselective method should not be undertaken unless the native type and topological isomer can be separated [19,20]. The results reported herein demonstrate that the stepwise disulfide formation strategy for Cys-rich peptides can provide not only a native form but also kinetically trapped topological isomer. Importantly, only the topological isomer was obtained when the C2–C5 disulfide bond of STa was formed in the last step [8], suggesting that the structure of its Acm2-precursor peptide and/or the order of disulfide formation leads to the folding of disulfide-rich peptides in the case of a chemical synthesis. Thus, chemical synthesis provides the possibility of preparing not only the native type but also the topological isomer, which still possesses biological activity. This topological isomer promises to provide new strategies for the use of peptides, such as in the preparation of vaccines.

4. Materials and Methods

4.1. Materials and Apparatus All chemicals and solvents were reagent grade unless otherwise described. The HPLC apparatus consisted of a Waters M600 multisolvent delivery system (Bedford, MA, USA) equipped with a Hitachi L-3000 detector and a D-2500 chromato-integrator or a LaChrom Elite system equipped with an L-2400 detector and a D-2500 chromato-integrator (Hitachi High-Technologies Corporation, Tokyo, Japan).

4.2. Syntheses of Peptide

The synthesis of the protected STh(6–18) peptide was performed manually by the solid-phase method, as described previously [8,9,21]. Briefly, protecting groups for functional groups were used, and the protected peptide resins were treated with anhydrous hydrogen fluoride (HF) in the presence of anisole at 0 ◦C for 60 min, as previously described [8], to remove the protecting groups except Molecules 2020, 25, 4798 9 of 11 for the Acm groups at given Cys residues (Figure S1) [8]. After drying in vacuo, the resulting residues were air-oxidized in 0.5 M AcONH4 buffer (pH 8.0) for two days to randomly form two disulfide bonds. The target peptide with the correct two disulfide bonds and 2 Cys(Acm) residues, 7,15 × ([Cys(Acm) ,2SS]-STh(6–18)), was predominantly produced and purified by HPLC, as described below [8].

4.3. I -Oxidation of the Peptide with Two Disulfide Bonds and 2 Cys(Acm) 2 × To investigate the effect of solvent on the folding of STa in the chemical synthesis, 7,15 [Cys(Acm) ,2SS]-STh(6–18) (a precursor peptide with two disulfide bonds) was treated with iodine to form a third disulfide bond under the several sets of conditions, as summarized in Table1. Briefly, the purified precursor peptide (5 10 4 M) was dissolved in 20% MeOH or other organic solvent, × − the resulting solution was dropped into the same volume of 20 10 3 MI in 80% MeOH or other organic × − 2 solvents containing 0.2 M HCl, and it was allowed to stand for 30 min at room temperature [8,9,15]. The reaction was stopped by adding the L-ascorbic acid saturated aqueous solution. The reaction product was directly purified by HPLC, as described below [8].

4.4. Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) Peptides were separated by RP-HPLC using a Cosmosil 5C -AR-II column (4.6 150 mm, Nacalai 18 × tesque, Inc., Kyoto, Japan), as described below. The synthetic peptides were eluted with a linear gradient of 5–50% or 10–50% CH3CN in 0.05% trifluoroacetic acid with increasing concentrations of CH3CN of l.0%/min or 0.5%/min at a flow rate of 1.0 mL/min. Fractions of the eluates were monitored for absorbance at 220 nm and confirmed by MALDI-TOF/MS and amino acid analyses.

4.5. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) The molecular masses of peptides were determined by means of an AXIMA confidence spectrometer (SHIMADZU Co., Kyoto, Japan) in the positive ion mode. Mass spectrometric analyses of peptides were carried out in the linear or reflector modes using α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Co., Tokyo, Japan) as a matrix. In a typical run, the lyophilized peptide (ca. 0.1 nmol) was dissolved in 0.05% TFA/50% CH3CN (1 µL), mixed with 1 µL of a matrix solution (10 mg/mL), and air-dried on the sample plate for MALDI-TOF/MS.

4.6. Amino Acid Analysis

Peptides were treated with 5.7 M HCl (constant boiling) at 110 ◦C for 24 h in a sealed bottle using a Waters picoTAGTM Workstation (Waters Co., Tokyo, Japan). The resulting hydrolysates were taken to dryness in vacuo and dissolved in 20 mM citrate buffer (pH 2). Amino acid analyses were performed on a Hitachi L-2000 amino acid analyzer (Hitachi Ltd., Tokyo, Japan).

4.7. CD Measurement

CD spectra were recorded on a JASCO J820 spectropolarimeter at 25 ◦C. The peptides were dissolved in 20 mM sodium phosphate buffer (pH 6.5), 50% MeOH/0.1 M HCl, or 50% i-PrOH/0.1 M HCl.

4.8. NMR Measurement and Structure Calculation All NMR experiments were performed on a JNM-ECA800 spectrometer (JEOL RESONANCE Inc., Tokyo, Japan) at 25◦C. The topological isomer of STh(6–18) was dissolved in 20 mM sodium phosphate buffer prepared with D2O or a 90% H2O/10% D2O mixture at pH 6.5. The precursor peptide, 7,15 [Cys(Acm) , 2SS]-STh(6–18), was dissolved in a 50% CD3COOD/50% D2O or a 50% CD3COOH/10% D2O mixture at pH 3. The peptide concentration was adjusted to approximately 5 mM and was placed in a 5-mm NMR tube (Shigemi, Tokyo, Japan) for all NMR experiments. Assignment of proton resonances was achieved by using a series of 1D and 2D spectra, including 1H, DQF-COSY Molecules 2020, 25, 4798 10 of 11 and 1H-1H NOESY. The NOE distance constraints for peptides were derived from 1H-1H NOESY spectra with mixing times of 200 ms. All structure calculations were performed with the CNS program [22]. Structure optimization and energy minimization were achieved using a simulated annealing algorithm. The final 10 lowest energy structures were analyzed using the MOLMOL [23] and PROCHECK programs [24]. Structural statistics for the 10 structures are included in Table S1. Graphical representations were prepared using PyMOL (www.pymol.org). All of these structures have been deposited in the (PDB) (http://www.rcsb.org/pdb/) under the accession code 7CSS and 7D37.

Supplementary Materials: Figure S1: Scheme for synthesis of the topological isomer of STh(6–18) by the stepwise formation of disulfide bonds. Figure S2: RP-HPLC profiles of the reaction solutions at each step of stepwise regioselective formation of disulfide bonds. Figure S3: RP-HPLC profiles of the Acm2-precursor peptide after air-oxidation. Figure S4: RP-HPLC profiles of Acm2-precursor peptide incubated under several conditions. Figure S5: CD spectra of the native form, topological isomer, and Acm2-precursor peptide of STh(6–18). Figure S6: The superpositions of 10 lowest energy structures of the topological isomer and Acm2-precursor peptide of STh(6–18). Figure S7: The superpositions of 10 lowest energy backbone structures of Acm2-precursor peptide of STh(6–18). Table S1: Data collection and refinement statistics. Table S2: Hydrogen bonds in the backbone structure of the native form, topological isomer and Acm2-precursor peptide. Author Contributions: Conceptualization, S.S. and Y.H.; methodology, S.S. and Y.H.; validation, S.S. and Y.H.; formal analysis, S.S., M.F., T.O., M.G., H.T. and Y.H.; investigation, S.S., M.F., T.O., M.G., H.T. and Y.H.; resources, S.S. and Y.H.; data curation, S.S. and Y.H.; writing—original draft preparation, S.S.; writing—review and editing, S.S. and Y.H.; visualization, S.S.; supervision, S.S. and Y.H.; project administration, Y.H.; funding acquisition, Y.H. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported in part by Grants-in-Aid for Scientific Research (C) (No. 16K01925) (Y.H.). Acknowledgments: The authors thank Miho Fujii, Yuhei Itani, and Naoto Emura, (Faculty of Science and Engineering, Kindai University) for technical assistance in the early stage of this study. The authors thank Hironobu Hojo and Toru Kawakami (Division of Protein Organic Chemistry, Institute for Protein Research, Osaka University) for the use of HF apparatus and amino acid analysis in the peptide synthesis. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviation

7,15 2SS: two disulfide bonds; Acm, acetamidomethyl; Acm2-precursor, [Cys(Acm) ,2SS]-STh(6–18); CD, circular dichroism; COSY, correlation spectroscopy; GC-C, guanylyl cyclase-C; HF, hydrogen fluoride; NOESY, nuclear overhauser effect spectroscopy; RMSD, root mean square deviation; STa, heat-stable enterotoxin; THF, tetrahydrofuran.

References

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