Quinsey Noelene S, Fitton Hazel L, Coughlin Paul, Whisstock James C, Dafforn Timothy R, Carrell Robin W, Bottomley Stephen P, Pike Robert N
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Biochemistry. 2003 Sep 2;42(34):10169-73. doi: 10.1021/bi0349322.
The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.
丝氨酸蛋白酶抑制剂（serpins）的快门区域由一些高度保守的残基组成，这些残基对稳定性和功能都至关重要。该区域发生替代的几种抗凝血酶变体不稳定，使携带者易患血栓形成。尽管迄今为止研究的快门区域中的大多数突变对丝氨酸蛋白酶抑制剂的稳定性和功能有害，但α1-抗胰蛋白酶中苯丙氨酸51被亮氨酸替代会增强稳定性。在这里，我们研究了在抗凝血酶中引入类似突变（苯丙氨酸77突变为亮氨酸）的影响。该突变不影响与蛋白酶相互作用的动力学。然而，引人注目的是，该蛋白质的热稳定性明显降低，与野生型重组抗凝血酶相比，丝氨酸蛋白酶抑制剂的解链温度降低了13℃。进一步的研究表明，与野生型抗凝血酶不同，该突变体在轻度加热后会采用潜伏（无活性）构象。先前关于使抗凝血酶不稳定的快门区域突变的研究表明，此类变体对肝素五糖和全长肝素均具有增强的亲和力。N135A/F77L突变体对肝素五糖的亲和力不变，但对全长肝素的亲和力增加。我们认为，苯丙氨酸77突变为亮氨酸的突变会导致抗凝血酶中D螺旋顶部周围的构象变化，特别是可能介导额外抗凝血酶/肝素相互作用的精氨酸132和133残基。本文还证明了抗凝血酶和α1-抗胰蛋白酶的快门区域之间存在主要差异，因为抗胰蛋白酶中的稳定突变在抗凝血酶中具有相反的作用。
