Jagger, Alistair M; (2019) Biophysical characterisation of structural dynamics in wild type and disease causing mutants of Alpha-1 antitrypsin using NMR approaches. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Alpha-1 Antitrypsin (α1AT) is a well-characterised member of the serine protease inhibitor (serpin) superfamily. As a 52kDa glycosylated protein, α1AT is involved in the regulation of proteolytic cascades in the body. Mutations, such as Z (E342K) and S (E264V) induce α1AT to aggregate into long chains called polymers at the site of synthesis in the endoplasmic reticulum of hepatocytes. These toxic species cause liver cirrhosis and hepatocellular carcinoma, while the reduction in available protease inhibitor leads to emphysema and COPD. Polymerisation involves transition through a polymerogenic intermediate that is accessed from the native state of disease-causing variants. This transiently populated species holds the most promise to inform therapies that block polymerisation. Here we use solution state NMR of 1H, 13C methyl labelled α1AT to characterise the native state conformations at high resolution under near physiological conditions. We describe NMR-based and structure-based approaches for the assignment of 72% of the methyl spectrum of α1AT. Experiments that probe protein dynamics in native α1AT across a range of timescales reveal conformational exchange with an invisible state, while perturbing the molecule with temperature reveals a population accessing a higher energy state. To investigate the structural consequences of disease-causing mutations and glycosylation, we describe a novel approach using NMR at natural isotopic abundance to study α1AT purified directly from patients. We show for the first time, at residue-specific resolution, that the native state conformation of Z α1AT is different to that of WT α1AT, notwithstanding recent contradictory evidence from X-ray crystal structures of this mutant. Using a small molecule selective for Z α1AT we show that this mutant populates a higher energy state conformation to approximately 20% at 298K. Finally we describe the characterisation of the mechanism of action of an antibody that is capable of blocking α1AT polymerisation. Antibody binding to an allosteric site in helix A induces an ‘open’ conformation in β-sheet A, as evidenced by increased peptide incorporation and destabilisation of the serpin:enzyme complex, which, out-of-step with other dynamic changes, is sufficient to inhibit polymerisation.

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