Stevens R C, Gouaux J E, Lipscomb W N
Gibbs Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138.
Biochemistry. 1990 Aug 21;29(33):7691-701. doi: 10.1021/bi00485a019.
The crystal structure of Escherichia coli aspartate carbamoyltransferase complexed with adenosine 5'-triphosphate (ATP) has been solved by molecular replacement and has been refined to a crystallographic residual of 0.17 at 2.6-A resolution by using the computer program X-PLOR. The unit cell dimensions of this crystal form are a = b = 122.2 A and c = 143.3 A and the space group is P321. Although the c-axis unit cell dimension is approximately 1 A longer than the corresponding dimension of the CTP-ligated P321 crystal form (c = 142.2 A), the ATP-ligated enzyme adopts a T-like quaternary structure. The base moiety of ATP interacts with residues Glu10, Ile12, and Lys60 while the ribose is near Asp19 and Lys60; the triphosphate entity is bound to Lys94, although His20 and Arg96 are nearby. We observe a higher occupancy for ATP in the allosteric site of the R1 regulatory chain in comparison to the occupancy of the R6 allosteric site. These crystallographically independent sites are related by a molecular 2-fold axis. There are other violations of the noncrystallographic symmetry that are similar to those observed in the refined CTP-ligated aspartate carbamoyltransferase structure. These infringements on the molecular symmetry might be the result of intermolecular interactions in the crystal. To ensure the most meaningful comparison with the ATP-ligated structure, we refined the previously reported CTP-bound and unligated structures to crystallographic residuals between 0.17 and 0.18 using X-PLOR. These X-PLOR refined structures are not significantly different from the initial structures that had been crystallographically refined by a restrained least-squares method. After making all possible comparisons between the CTP- and ATP-ligated and the unligated T-state structures, we find that the most significant differences are located at the allosteric sites and in small changes in the quaternary structures. At the allosteric site, the binding of CTP and ATP successively enlarges the nucleotide binding cavity, particularly in the vicinity of the base. The changes in the quaternary structure can be characterized by an increase in the separation of the catalytic trimers by approximately 0.5 A as ATP binds to the unligated T structure. On the basis of these structural studies, we discuss the relationships between the conformational differences in the allosteric site and the small changes in the quaternary structure within the T form to the possible mechanisms for CTP inhibition and ATP activation.
通过分子置换法解析了与三磷酸腺苷（ATP）结合的大肠杆菌天冬氨酸氨甲酰基转移酶的晶体结构，并使用计算机程序X-PLOR在2.6埃分辨率下将其精修至晶体学残余因子为0.17。该晶体形式的晶胞尺寸为a = b = 122.2埃，c = 143.3埃，空间群为P321。尽管c轴晶胞尺寸比CTP连接的P321晶体形式的相应尺寸（c = 142.2埃）长约1埃，但ATP连接的酶采用类T四级结构。ATP的碱基部分与Glu10、Ile12和Lys60残基相互作用，而核糖靠近Asp19和Lys60；三磷酸实体与Lys94结合，尽管His20和Arg96在附近。我们观察到，与R6变构位点的占有率相比，ATP在R1调节链变构位点的占有率更高。这些晶体学上独立的位点通过分子二重轴对称相关。还有其他违反非晶体学对称性的情况，类似于在精修的CTP连接的天冬氨酸氨甲酰基转移酶结构中观察到的情况。这些对分子对称性的侵犯可能是晶体中分子间相互作用的结果。为了确保与ATP连接结构进行最有意义的比较，我们使用X-PLOR将先前报道的CTP结合和未结合结构精修至晶体学残余因子在0.17至0.18之间。这些经X-PLOR精修的结构与通过约束最小二乘法进行晶体学精修的初始结构没有显著差异。在对CTP和ATP连接以及未结合的T态结构进行所有可能的比较之后，我们发现最显著的差异位于变构位点以及四级结构的微小变化中。在变构位点，CTP和ATP的结合依次扩大了核苷酸结合腔，特别是在碱基附近。当ATP与未结合的T结构结合时，四级结构的变化可以通过催化三聚体之间的间距增加约0.5埃来表征。基于这些结构研究，我们讨论了变构位点的构象差异和T形式内四级结构的微小变化与CTP抑制和ATP激活的可能机制之间的关系。
