Lowen, Sarah Min; (2025) Structural investigation of pathogenic conformational change and allosteric networks in alpha-1 antitrypsin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The glycoprotein alpha-1 antitrypsin (AAT) is a 52 kDa serine protease inhibitor (serpin) found at high concentrations in human plasma. The Z mutation (E342K) promotes both misfolding and ordered aggregation. This leads to accumulation of AAT polymers in the endoplasmic reticulum of hepatocytes and subsequent low levels of circulating functional AAT in plasma. This can cause liver cirrhosis and early onset emphysema associated with AAT deficiency (AATD). In this study, NMR spectra of heat-induced 13C-methyl-labelled polymers and 1H methyl spectra of liver-derived polymers were found to show equivalence to that of AAT in a post-protease-encounter conformation. This resonance equivalence could only be explained by a mechanism in which there is full insertion of a region known as the ‘reactive centre loop’ and an intermolecular domain swap of the C-terminus of the molecule. These data explain the mechanism of aberrant polymer formation in Z AAT deficiency. In hepatocytes, interactions between polymers result in condensates, known as ‘inclusion bodies’. Natural isotopic abundance 2D solid-state NMR spectra of inclusion bodies isolated from the liver tissue of individuals homozygous for the Z allele showed a resonance distribution that indicates inclusion body aggregates remain intact and properly folded in situ. Central to serpin polymerisation and function is its ability to undergo extensive conformational change, a process that lends itself to allosteric regulation. Solution-state NMR and X-ray crystallography undertaken here revealed that binding by bivalent zinc and copper ions can result in allosteric structural rearrangement, increased global flexibility, and increased polymerisation rates. These data allow definition of the structural changes that drive polymer formation and the nature of the inter-subunit polymer linkage. This study has also revealed for the first time the potential for allosteric effects on AAT by metal binding, and a methodological basis for the study of tissue-derived inclusion bodies by solid-state NMR.

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