title: Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway.
abstract: The common severe Z mutation (E342K) of α1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular 'snapshots', the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1', resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol(-1).
authors: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
authors: Imran
authors: I
authors: Haq
authors: James A
authors: JA
authors: Irving
authors: Sarah V
authors: SV
authors: Faull
authors: Jennifer A
authors: JA
authors: Dickens
authors: Adriana
authors: A
authors: Ordóñez
authors: Didier
authors: D
authors: Belorgey
authors: Bibek
authors: B
authors: Gooptu
authors: David A
authors: DA
authors: Lomas
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D058105:Polymerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin
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