Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band3

Band 3(B3),theaniontransporter,isanintegralmembraneproteinthatplaysakeystructuralroleby anchoringtheplasmamembranetothespectrin-basedmembraneskeletonintheredcell.Inaddition, it alsoplaysacriticalroleintheassemblyofglycolyticenzymestoregulateredcellmetabolism. However,itsabilitytorecruitproteinsthatcanpreventmembraneoxidationhasnotbeenpreviously explored.Inthisstudy,usingavarietyofexperimentalapproachesincludingcross-linkingstudies, fluorescenceanddichroicmeasurements,surfaceplasmonresonanceanalysis,andproteolyticdigestion assays,wedocumentthattheantioxidantproteinperoxiredoxin-2(PRDX2),thethirdmostabundant cytoplasmicproteininRBCs,interactswiththecytoplasmicdomainofB3.Thesurfaceelectrostatic potentialanalysisandstoichiometrymeasurementsrevealedthattheN-terminalpeptideofB3is involvedintheinteraction.PRDX2underwentaconformationalchangeuponitsbindingtoB3without losingitsperoxidaseactivity.HemichromeformationinducedbyphenylhydrazineofRBCsprevented membraneassociationofPRDX2,implyingoverlappingbindingsites.Documentationoftheabsenceof bindingofPRDX2toB3Neapolisredcellmembranes,inwhichtheinitialN-terminal11aminoacidsare deleted,enabledustoconcludethatPRDX2bindstotheN-terminalcytoplasmicdomainofB3andthat the first11aminoacidsofthisdomainarecrucialforPRDX2membraneassociationinintactRBCs. ThesefindingsimplyyetanotherimportantroleforB3inregulatingredcellmembranefunction.