Peptide Binding Sites of Connexin Proteins

Ágnes Simon,Julianna Kardos,László Héja,Csaba Magyar

doi:10.3390/chemistry2030042

Abstract

Intercellular gap junction (GJ) contacts formed by the coupling of connexin (Cx) hemichannels (HCs) embedded into the plasma membranes of neighboring cells play significant role in the development, signaling and malfunctions of mammalian tissues. Understanding and targeting GJ functions, however, calls for finding valid Cx subtype-specific inhibitors. We conjecture the lack of information about binding interactions between the GJ interface forming extracellular EL1 and EL2 loops and peptide mimetics designed to specifically inhibit Cx43HC coupling to Cx43GJ. Here, we explore active spots at the GJ interface using known peptide inhibitors that mimic various segments of EL1 and EL2. Binding interactions of these peptide inhibitors and the non-peptide inhibitor quinine has been modelled in combination with the use of blind docking molecular mechanics (MM). The neuron-specific Cx36HC and astrocyte-specific Cx43HC subtypes were modelled with a template derived from the high-resolution structure of Cx26GJ. GJ-coupled and free Cx36HC and Cx43HC models were obtained by dissection of GJs (GJ-coupled) followed by 50 ns molecular dynamics (free). Molecular mechanics (MM) calculations were performed by the docking of inhibitors, explicitly the designed Cx43 EL1 or EL2 loop sequence mimetics (GAP26, P5 or P180–195, GAP27, Peptide5, respectively) and the Cx36 subtype-specific quinine into the model structures. In order to explore specific binding interactions between inhibitors and CxHC subtypes, MM/Generalized Born Surface Area (MM/GBSA) ΔGbind values for representative conformers of peptide mimetics and quinine were evaluated by mapping the binding surface of Cx36HC and Cx43HC for all inhibitors. Quinine specifically contacts Cx36 EL1 residues V54-C55-N56-T57-L58, P60 and N63. Blocking the vestibule by the side of Cx36HC entry, quinine explicitly interacts with the non-conserved V54, L58, N63 residues of Cx36 EL1. In addition, our work challenges the predicted specificity of peptide mimetics, showing that the docking site of peptides is unrelated to the location of the sequence they mimic. Binding features, such as unaffected EL2 residues and the lack of Cx43 subtype-specificity of peptide mimetics, suggest critical roles for peptide stringency and dimension, possibly pertaining to the Cx subtype-specificity of peptide inhibitors.

Highlights

Gap junctions (GJs) formed by the coupling of various connexin (Cx) subtype hemichannels (CxHCs, connexons) maintain adhesion and conduction between adjacent cells [1,2,3]
Warner et al [8] conjectured HC coupling to GJ and conserved amino acid (AA) motifs, with QPG of EL1 being among the residues that possibly intervene HC coupling to GJ
The arrangement illustrates that permutations of the vertical inter-loop interface joined with the horizontal loop-periphery interface are beyond the most exposed EL1 loop sequences, lying on the front of the CxHC coupling reaction. These findings conclusively suggest that the location of interfaces identified by matching AA sequences of designed peptide mimetics argue against the notion that mfncoooirnmtmitoieignntiugcotshTucaMsotutohlmdecilhdmicaireenestncitctealslykfecionorthuhmilebdinisthgdcaoTiprnMeencoethlxfyeoalniivcnceehossiuttbiapbiktluienlcegtohbnteyonseGthhxJaeo.pnNseiodcoteoafuboaplfyvlH,ienEsCgtLi1ebtnusotelreqyGub

Summary

Introduction

Gap junctions (GJs) formed by the coupling of various connexin (Cx) subtype hemichannels (CxHCs, connexons) maintain adhesion and conduction between adjacent cells [1,2,3]. They are recognized as critical players in the development and disease of mammalian tissues [4,5,6] (and reference cited). One third of EL1 AAs (x) are less conserved, providing opportunity for subtype-specific inhibitor design. We hypothesize that Cx subtype-specific coupling of HC to GJ shall become true by undertaking the validation of the binding of mimetic peptides and quinine at the extracellular interface of Cx43HC and/or Cx36HC. BolyebculinladrdMocekcihnagnpicesp/tGideenseerqaulieznecde mBoimrnetSicusr, fwaceealAsorea (MeMxp/GloBreSdA)th∆eGvbailniddvbainludeins gwaerreeap. eCraflocrumlaetdio[n3s4o] fotnhtehMe 3o0lebcueslatrscMoericnhganpiocse/Gs eonfetrhaelibzleidndBodronckSiunrgfatcreials to AfitreCax(HMCMp/GepBtSiAde) ΔinGhbiinbdivtoarluseasnwdeqrue ipneirnfeo.rmThedis[m34e]tohnotdheha30s bpersotvsecdortiongbepoaspesoowfethrfeublltionodldtocpkrinedgict bintrdiainlsgtaoffifint iCtixeHs Canpdeipdteidnetiifnyhtihbeitocorsrraencdt bqiunidniinnge.pTohsiessmfoerthpordotheains–ppreopvteiddetocobme pa lpeoxwese[r3fu5]l.tool to predict binding affinities and identify the correct binding poses for protein–peptide complexes [35]

Results and Discussion

Determination of the Position of TM Regions

Conclusions