Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

The common severe Z mutation (E342K) of α(1)-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 α(1)-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P(14)P(12) and P(4), but not P(10)P(8) or P(2)P(1′), 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 P(14)P(12) region self-inserts early during activation, while the interaction between P(6)P(4) and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P(6)P(4), but not P(14)P(12), yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol(−1).