Targeting a surface cavity of alpha 1-antitrypsin to prevent conformational disease.
Conformational diseases are caused by a structural rearrangement within a protein that results in aberrant intermolecular linkage and tissue deposition. This is typified by the polymers that form with the Z deficiency variant of alpha 1-antitrypsin (Glu-342 --> Lys). These polymers are retained within hepatocytes to form inclusions that are associated with hepatitis, cirrhosis, and hepatocellular carcinoma. We have assessed a surface hydrophobic cavity in alpha1-antitrypsin as a potential target for rational drug design in order to prevent polymer formation and the associated liver disease. The introduction of either Thr-114 --> Phe or Gly-117 --> Phe on strand 2 of beta-sheet A within this cavity significantly raised the melting temperature and retarded polymer formation. Conversely, Leu-100 --> Phe on helix D accelerated polymer formation, but this effect was abrogated by the addition of Thr-114 --> Phe. None of these mutations affected the inhibitory activity of alpha 1-antitrypsin. The importance of these observations was underscored by the finding that the Thr-114 --> Phe mutation reduced polymer formation and increased the secretion of Z alpha 1-antitrypsin from a Xenopus oocyte expression system. Moreover cysteine mutants within the hydrophobic pocket were able to bind a range of fluorophores illustrating the accessibility of the cavity to external agents. These results demonstrate the importance of this cavity as a site for drug design to ameliorate polymerization and prevent the associated conformational disease.
Parfrey
Helen
H
Respiratory Medicine Unit, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom. hp226@cam.ac.uk
Mahadeva
Ravi
R
Ravenhill
Neil A
NA
Zhou
Aiwu
A
Dafforn
Timothy R
TR
Foreman
Richard C
RC
Lomas
David A
DA
D000818:Animals; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002918:Chymotrypsin / Q000737:chemistry; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D005355:Fibrosis / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006505:Hepatitis / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014981:Xenopus; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000378:metabolism
D011108:Polymers; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D013912:Threonine; D010649:Phenylalanine; D011189:Potassium Chloride; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D003545:Cysteine; D005998:Glycine