Prasad G S, Stura E A, McRee D E, Laco G S, Hasselkus-Light C, Elder J H, Stout C D
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037-1093, USA.
Protein Sci. 1996 Dec;5(12):2429-37. doi: 10.1002/pro.5560051205.
We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E. coli dUTPase. In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.
我们已经确定了猫免疫缺陷病毒（FIV）的dUTP焦磷酸酶（dUTPase）在1.9埃分辨率下的晶体结构。该结构通过使用P6(3)晶型的多重同晶置换（MIR）方法解析得到。结果表明，该酶是由14.3 kDa亚基组成的三聚体，与大肠杆菌dUTPase具有显著的结构相似性。在这两种酶中，反平行β-桶的C末端链参与相邻亚基的β-折叠，形成相互交错的、具有生物学功能的三聚体。在P6(3)晶型中，一个三聚体堆积在6(3)螺旋轴上，另一个堆积在三重轴上，因此每个不对称单元中有两个独立的单体。每个三聚体的三重轴上的三个天冬氨酸残基与一个Mg2+离子配位。17种病毒、原核生物和真核生物dUTPase序列的比对揭示了五个保守基序。其中四个位于亚基对之间的界面上，定义了一个假定的活性位点区域；第五个位于C末端的16个残基中，在晶体中是无序的。来自所有三个亚基的保守基序是形成给定活性位点所必需的。关于病毒蛋白表达，特别有趣的是，dUTPase（DU）基因位于逆转录病毒基因组的酶盒Pol基因的中间。Pol多蛋白中编码的其他酶，包括蛋白酶（PR）、逆转录酶（RT）以及很可能的整合酶（IN），都是二聚体酶，这意味着活性三聚体dUTPase的表达化学计量与其他Pol编码的酶不同。此外，由于结构限制，dUTPase在从多蛋白切割之前不太可能达到活性形式。
