Ordered Self-Assembly Mechanism of a Spherical Oncoprotein Oligomer Triggered by Zinc Removal and Stabilized by an Intrinsically Disordered Domain 英文参考文献.docVIP

OrderedSelf-AssemblyMechanismofaSpherical OncoproteinOligomerTriggeredbyZincRemovaland StabilizedbyanIntrinsicallyDisorderedDomain ClaraSmal1,LeonardoG.Alonso2,DianaE.Wetzler3,AngelesHeer1,GonzalodePratGay1* 1Fundacio′n Instituto Leloir and Instituto de Investigaciones Bioqu?′micas-Conicet, Buenos Aires, Argentina, 2XBio Inc., Buenos Aires, Argentina, 3Departamento de Qu?′micaBiolo′gica,FacultaddeCienciasExactasyNaturales,UniversidaddeBuenosAires,CiudadUniversitaria,BuenosAires,Argentina Abstract Background:Self-assemblyisacommonthemeinproteinsofunrelatedsequencesorfunctions.Thehumanpapillomavirus E7oncoproteinisanextendeddimerwithanintrinsicallydisordereddomain,thatcanformlargesphericaloligomers.These arethemajorspeciesinthecytosolofHPVtransformedandcancerouscells.E7bindstoalargenumberoftargets,someof whichleadtocelltransformation.Thus,theassemblyprocessnotonlyisofbiologicalrelevance,butrepresentsamodel systemtoinvestigateawidelydistributedmechanism. Methodology/PrincipalFindings:Usingvarioustechniques,wemonitoredchangesinsecondary,tertiaryandquaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formationofamonomeric‘‘Z-nucleus’’afterzincremoval,followedbyanelongationphaseconsistingofsequentialsecond- order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 mM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a ‘‘conformation editing’’ mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsicallydisordereddomainstabilizestheoligomer,preventinganinsolubleamyloidroute. Conclusion: We described a controlled ordered mecha