X-ray structural analysis of tryptophan synthase a and P-subunits and its α_2β_2 complex from hyperthermophile

本项目测定了极端嗜热菌（Pyrococcus furiosus）色氨酸合成酶α-亚基、β_2-亚基和α_2β_2复合体的X射线结构，以阐明α_2β_2复合体形成时亚基相互作用相互激活的分子基础和极端嗜热菌蛋白质的稳定机制。在此期间，我们成功地确定了2.0 A处的α亚基和2.3 A处的β_2亚基的结构，但在3.2 A以上的分辨率下，未能确定复合物的结构。本文对α亚基的研究结果进行了总结。虽然鼠伤寒沙门氏菌色氨酸合酶α_2β_2复合体的结构已被确定，但这是第一次单独报道α亚基的结构。与S. furiosus相比，Pf-α-亚基的N端和C端分别缺少12个和6个残基，两个环区各缺少1个残基。鼠伤寒沙门氏菌（St-α-亚基），导致N-末端螺旋缺失和C-末端螺旋缩短。Pf-α-亚基的结构与α_2β_2复合物中St-α-亚基的结构基本相似。两种结构之间的差异进行了讨论，与较高的稳定性的Pf-α-亚基和复合物的形成的α和β-亚基。量热结果表明Pf-α-亚基具有极高的热稳定性，其高稳定性是由熵效应引起的。基于两种蛋白质的结构信息，我们分析了每个稳定因子的贡献，可以得出结论，蛋白质内部的疏水相互作用并不有助于Pf-α-亚基的更高稳定性。相反，离子对的增加、空腔体积的减小以及由于多肽链缩短而引起的熵效应在Pf-α-亚基的极高稳定性中起重要作用。

This project is to determine the X-ray structures of α-subunit, β_2-subunite, and α_2β_2 complex of tryptophan synthase from hyperthermophile, Pyrococcus furiosus, in order to elucidate the molecular basis of the mutual activation of the subunit interaction due to the formation of the α_2β_2 complex and the stabilization mechanism of proteins from hyperthermophile. In the period, we could succeed to determine the structures of a-subunit at 2.0 A and β_2-subunite at 2.3 A.However, the structure of the complex was not determined at higher resolutions than 3.2 A. In this report the results of α-subunit are summarized. Although the structure of the tryptophan synthase α_2β_2 complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the α-subunit alone. The α-subunit from P. furiosus (Pf-α-subunit) lacked 12 and 6 residues at the N- and C-termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-α-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-α-subunit was essentially similar to that of the St-α-subunit in the α_2β_2 complex. The differences between both structures were discussed in connection with the higher stability of the Pf-α-subunit and the complex formation of the α and β-subunits. Calorimetric results indicated that the Pf-α-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-α-subunit. Rather the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening the polypeptide chain play important roles in extremely high stability in Pf-α-subunit.