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 polymerisation pathway. Despite these molecular 'snapshots', the details of the transition between monomer and polymer remain only partially understood. We surveyed the 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 polymerisation of the Z variant, consistent with polymerisation from a near-native conformation. We have developed a Förster resonance energy transfer (FRET)-based assay to monitor polymerisation in small sample volumes. An in vitro assessment revealed position-specific effects on the unimolecular and multimolecular phases of polymerisation: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and β-sheet A presents a kinetic barrier late in the polymerisation pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 kJ mol-1 to ~550 kJ mol-1.
Original language | English |
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Journal | Bioscience Reports |
DOIs | |
Publication status | E-pub ahead of print - 9 May 2013 |