ABSTRACT
The common Z mutant (Glu342Lys) of alpha(1)-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native alpha(1)-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 A structure of Thr114Phe alpha(1)-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-beta-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z alpha(1)-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of alpha(1)-antitrypsin and increase secretion of monomeric protein in cell models of disease.
Subject(s)
alpha 1-Antitrypsin/chemistry , Amino Acid Substitution , Animals , COS Cells , Chlorocebus aethiops , Crystallography, X-Ray , Drug Design , Female , Hepatocytes/metabolism , Humans , In Vitro Techniques , Models, Molecular , Mutation, Missense , Oocytes/metabolism , Protein Conformation , Protein Folding , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Xenopus , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolismABSTRACT
The Z mutant of alpha1-antitrypsin (Glu342Lys) causes a domain swap and the formation of intrahepatic polymers that aggregate as inclusions and predispose the homozygote to cirrhosis. We have identified an allosteric cavity that is distinct from the interface involved in polymerization for rational structure-based drug design to block polymer formation. Virtual ligand screening was performed on 1.2 million small molecules and 6 compounds were identified that reduced polymer formation in vitro. Modeling the effects of ligand binding on the cavity and re-screening the library identified an additional 10 compounds that completely blocked polymerization. The best antagonists were effective at ratios of compound to Z alpha1-antitrypsin of 2.5:1 and reduced the intracellular accumulation of Z alpha1-antitrypsin by 70% in a cell model of disease. Identifying small molecules provides a novel therapy for the treatment of liver disease associated with the Z allele of alpha1-antitrypsin.
