Crystallographic and cellular characterisation of two mechanisms stabilising the native fold of alpha1-antitrypsin: implications for disease and drug design.
Crystallographic and cellular characterisation of two mechanisms stabilising the native fold of alpha1-antitrypsin: implications for disease and drug design.
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两种机制的晶体学和细胞表征稳定α1-抗胰蛋白酶的天然折叠：对疾病和药物设计的影响。
DOI：
10.1016/j.jmb.2009.01.069
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发表时间：
2009-04-10
影响因子：
5.6
通讯作者：
Barrett, Tracey E.
中科院分区：
文献类型：
--
作者：
Gooptu, Bibek;Miranda, Elena;Nobeli, Irene;Mallya, Meera;Purkiss, Andrew;Brown, Sarah C. Leigh;Summers, Charlotte;Phillips, Russell L.;Lomas, David A.;Barrett, Tracey E.
α1-抗胰蛋白酶的常见 Z 突变体 (Glu342Lys) 会导致保留在肝细胞内的聚合物的形成。这会导致肝脏疾病，而血浆中缺乏一种重要的蛋白酶抑制剂则容易导致肺气肿。 Thr114Phe 和 Gly117Phe 突变与表面空腔接壤，被确定为合理药物设计的目标。这些突变保留了抑制活性，但在体外减少了野生型天然 α1-抗胰蛋白酶的聚合，并增加了非洲爪蟾卵母细胞疾病模型中的分泌。为了了解这些效应，我们结晶了两种突变体并解析了它们的结构。 Thr114Phe α1-抗胰蛋白酶的 2.2 Å 结构表明，突变的影响完全是由明确的部分空腔阻断介导的，并允许通过计算机筛选能够模拟突变影响的片段。 Gly117Phe 突变的运作方式不同，在螺旋 F-β-折叠 A 界面中重新包装芳香族侧链，从而诱导相邻 F 螺旋的半圈向下移动。我们进一步表征了这两种突变与 Z 突变相结合在疾病真核细胞模型中的作用。两种突变都增加了天然构象中 Z α1-抗胰蛋白酶的分泌，但双突变体仍然比野生型 (M) 蛋白更具聚合性。总而言之，这些数据支持 Thr114Phe 和 Gly117Phe 突变稳定 α1-抗胰蛋白酶的天然折叠并增加疾病细胞模型中单体蛋白分泌的不同机制。
The common Z mutant (Glu342Lys) of α1-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 α1-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 α1-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 α1-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 α1-antitrypsin and increase secretion of monomeric protein in cell models of disease.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.9
作者：
James, EL;Bottomley, SP
通讯作者：
Bottomley, SP
DOI：
10.1073/pnas.97.1.67
发表时间：
2000-01-04
影响因子：
11.1
作者：
Gooptu, B;Hazes, B;Lomas, DA
通讯作者：
Lomas, DA
影响因子：
2.9
作者：
Cabrita, LD;Whisstock, JC;Bottomley, SP
通讯作者：
Bottomley, SP
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL