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Effect of Conformational Diversity on the Bioactivity of Μ-Conotoxin PIIIA Disulfide Isomers

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Effect of Conformational Diversity on the Bioactivity of Μ-Conotoxin PIIIA Disulfide Isomers marine drugs Article Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers 1, 1, 2, 1 Ajay Abisheck Paul George y, Pascal Heimer y, Enrico Leipold y, Thomas Schmitz , Desiree Kaufmann 3, Daniel Tietze 3 , Stefan H. Heinemann 4 and Diana Imhof 1,* 1 Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany 2 Department of Anesthesiology and Intensive Care, University of Lübeck, Ratzeburger Allee 160, D-23562 Lübeck, Germany 3 Eduard Zintl Institute of Inorganic and Physical Chemistry, Darmstadt University of Technology, Alarich-Weiss-Str. 4, D-64287 Darmstadt, Germany 4 Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, D-07745 Jena, Germany * Correspondence: [email protected]; Tel.: +49-228-73-5254 These authors contributed equally to this work. y Received: 5 June 2019; Accepted: 25 June 2019; Published: 2 July 2019 Abstract: Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer’s activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble. Keywords: µ-conotoxin; PIIIA; voltage-gated sodium channel; disulfide connectivity; peptide folding; electrophysiology; molecular docking; molecular dynamics 1. Introduction Covalent linkage of cysteine residues by disulfide bonds is fundamental for the folding, stability, and function of many peptides and proteins [1–4]. The formation of correct disulfide linkages is particularly important for peptides and proteins with higher-order disulfide networks (>2 disulfide bonds) because incorrectly assembled disulfide bonds may directly interfere with the biological functions of these molecules. The venom of marine cone snails is a rich source of small- to medium-sized disulfide-stabilized peptides, so-called conotoxins, most of which specifically target ion channels and receptors in the membranes of excitable cells [5]. Conotoxins are recognized for their pharmacological and therapeutic Mar. Drugs 2019, 17, 390; doi:10.3390/md17070390 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 390 2 of 18 potentialMar. andDrugs they2019,, 17 are,, xx FORFOR models PEERPEER REVIEWREVIEW to study the impact of disulfide bonds on the structural stability2 of 19 of Mar. Drugs 20192019,, 1717,, xx FORFOR PEERPEER REVIEWREVIEW 22 ofof 1919 Mar. Drugs 2019, 17, x FOR PEER REVIEW 2 of 19 disulfide-richMar. Drugs peptides2019, 17, x FOR and PEER proteins REVIEW [ 4,6–8]. For example, it was shown that the biological activities2 of 19 Mar.Mar. DrugsDrugs 20192019,, 1717,, xx FORFOR PEERPEER REVIEWREVIEW 22 ofof 1919 Mar.therapeutic Drugs 2019 potential,, 17,, xx FORFOR andPEERPEER they REVIEWREVIEW are models to study the impact of disulfide bonds on the structural2 of 19 and receptortherapeutictherapeutic specificities potentialpotential of andand some theytheyµ -, areareµO-, models and αto-conotoxins study the impact changed of disulfide significantly bonds if on the the native structural disulfide therapeuticstabilitytherapeutic of disulfide-rich potentialpotential andand peptides theythey areare and modelsmodels proteins toto studystudy [4,6– 8]. thethe For impactimpact example, ofof disulfidedisulfide it was shown bondsbonds that onon thethe biologicalstructuralstructural patternstherapeuticstabilitystabilitytherapeutic of the ofof peptides disulfide-rich disulfide-rich potentialpotential were andand peptidespeptides experimentallytheythey areare and and modelsmodels proteinsproteins to modifiedto studystudy [4,6[4,6– 8].thethe [ 9For –impactimpact12α example,]. ofof disulfidedisulfide it was shown bondsbonds that onon thethe structuralbiologicalstructural therapeuticactivitiesstability ofand disulfide-richpotential receptor and specificities peptides they are and modelsof someproteins to µ-, study [4,6µO-,– the8]. and For impact α example,-conotoxins of disulfide it was changed shown bonds significantly thaton the the structuralbiological if the stabilityactivitiesstability ofof and disulfide-richdisulfide-rich receptor specificities peptidespeptides andand of proteinssomeproteins µ-, [4,6[4,6 µO-,–8]. and For α example,-conotoxins it was changed shown significantly that the biological if the Thestabilityactivitiesnativestability 3-disulfide-bonded disulfide ofof and disulfide-richdisulfide-rich receptor patterns specificitiesµ peptidesofpeptides-conotoxins the peptid andand of es proteinssomeproteins specifically were µ-, experimentally [4,6 [4,6µO-,–– antagonize8].8]. and ForFor αexample,example,-conotoxins modified voltage-gated itit waswas [9changed– 12]. shownshown sodium significantly thatthat thethe channels biologicalbiological if the (Na V activitiesstabilitynativeactivities disulfide of andand disulfide-rich receptorreceptor patterns specificitiesspecificities peptidesof the peptid and ofof es proteinssomesome were µ-,µ-, experimentally [4,6 µO-,µO-,–8]. andand For α example,-conotoxins-conotoxins modified it was [9 changedchanged– 12].shown significantly significantlythat the biological ifif thethe activitiesnativeactivities disulfide andand receptorreceptor patterns specificitiesspecificities of the peptid ofof es somesome were µ-,µ-, experimentally µO-,µO-, andand αα-conotoxins-conotoxins modified [9 changedchanged–12]. significantlysignificantly ifif thethe channels)activitiesnative makingThe disulfide 3-disulfide-bondedand them receptor patterns attractive specificities of the researchµ-conotoxins peptid of es toolssome were specifically asµ-, experimentally well µO-, as and potentialantagonize α-conotoxins modified pharmacological voltage-gated [9changed–12]. significantly sodium lead structures channels if the[ 13]. nativeThe disulfide 3-disulfide-bonded patterns of the µ-conotoxins peptides were specifically experimentally antagonize modified voltage-gated [9–12]. sodium channels µ native(NanativeV Thechannels) disulfidedisulfide 3-disulfide-bonded patternsmakingpatterns ofthemof thethe µ-conotoxins attractivepeptidpeptideses were wereresearch specifically experimentallyexperimentally tools antagonizeas well modifiedmodified as potentialvoltage-gated [9[9––12].12]. pharmacological sodium channels lead -Conotoxinsnative(NaVThe channels) disulfide are 3-disulfide-bonded so-called patternsmaking pore ofthem blockersthe µ-conotoxins attractivepeptid becausees were research specifically theyexperimentally tools bind antagonize toas thewell modified extracellular as voltage-gatedpotential [9–12]. pore pharmacological vestibulesodium channels and, lead hence, (Na(NaVVThe Thechannels)channels) 3-disulfide-bonded3-disulfide-bonded makingmaking themthem µ-conotoxins µ-conotoxins attractiveattractive researchresearch specificallyspecifically toolstools antagonizeantagonizeasas wellwell asas voltage-gatedpotentialvoltage-gatedpotential pharmacologicalpharmacological sodiumsodium channelschannels leadlead structures(NaVThe channels) 3-disulfide-bonded [13]. µ-Conotoxins making them µ-conotoxinsare attractive so-called+ research porespecifically blockers tools antagonize asbecause well asthey voltage-gatedpotential bind to pharmacologicalthe extracellularsodium channels porelead obstruct(Nastructures theV Thechannels) permeation 3-disulfide-bonded [13]. µ-Conotoxinsmaking of sodium them µ-conotoxinsare ionsattractive so (Na-called )research through porespecifically blockers tools the channels antagonizeas because well as [they14 voltage-gatedpotential]. Whilebind to some pharmacologicalthe extracellularsodiumµ-conotoxins, channels porelead such structuresstructures(NaV channels) [13].[13]. µ-Conotoxins µ-Conotoxinsmaking them areare attractive soso-called-called research porepore blockersblockers tools asbecausebecause well astheythey potential bindbind toto pharmacologicalthethe extracellularextracellular poreporelead (Navestibule(NaVV channels)channels) and, hence, makingmaking obstruct themthem theattractiveattractive permeation researchresearch of sodium toolstools asasions wellwell (Na asas+) throughpotentialpotential the pharmacologicalpharmacological channels [14]. While leadlead as µ-GIIIA(NavestibulestructuresV arechannels) veryand, [13]. specifichence, µ-Conotoxinsmaking obstruct for them skeletal aretheattractive sopermeation muscle-called
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