Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of α₁-Antitrypsin: Implications for Disease and Drug Design

TY - JOUR

T1 - Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of α1-Antitrypsin

T2 - Implications for Disease and Drug Design

AU - Gooptu, Bibek

AU - Miranda, Elena

AU - Nobeli, Irene

AU - Mallya, Meera

AU - Purkiss, Andrew

AU - Leigh Brown, Sarah C.

AU - Summers, Charlotte

AU - Phillips, Russell L.

AU - Lomas, David A.

AU - Barrett, Tracey E.

PY - 2009/4/10

Y1 - 2009/4/10

N2 - The common Z mutant (Glu342Lys) of α-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 α-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 Å structure of Thr114Phe α-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-β-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 α-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 α-antitrypsin and increase secretion of monomeric protein in cell models of disease.

AB - The common Z mutant (Glu342Lys) of α-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 α-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 Å structure of Thr114Phe α-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-β-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 α-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 α-antitrypsin and increase secretion of monomeric protein in cell models of disease.

U2 - 10.1016/j.jmb.2009.01.069

DO - 10.1016/j.jmb.2009.01.069

M3 - Article

AN - SCOPUS:62649174885

SN - 0022-2836

VL - 387

SP - 857

EP - 868

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 4

ER -