Bujacz G, Jaskólski M, Alexandratos J, Wlodawer A, Merkel G, Katz R A, Skalka A M
Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, MD 21702, USA.
Structure. 1996 Jan 15;4(1):89-96. doi: 10.1016/s0969-2126(96)00012-3.
Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes.
The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN.
Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.
结构相关的酶超家族成员，包括核糖核酸酶H（RNase H）、RuvC解离酶、MuA转座酶和逆转录病毒整合酶，其酶活性需要二价阳离子。到目前为止，仅在其中两种蛋白质（大肠杆菌和人类免疫缺陷病毒1型（HIV-1）核糖核酸酶H）的X射线晶体结构中报道了阳离子的位置。了解逆转录病毒整合酶活性位点中金属离子的放置细节对于理解这些酶的催化机制至关重要。
在存在二价阳离子（Mn2+或Mg2+）的情况下，已解析出禽肉瘤病毒整合酶（ASV IN）的酶活性催化结构域（第52至207位氨基酸残基）的结构，分辨率为1.7 - 2.2 Å。任一类型的单个离子与活性位点天冬氨酸的羧基相互作用，并利用四个水分子完成其八面体配位。天冬氨酸侧链和金属离子的放置与在该超家族的核糖核酸酶H成员中观察到的非常相似；然而，ASV IN活性位点中催化天冬氨酸的构象与HIV-1 IN中类似残基所报道的构象有显著差异。
所需金属离子的结合不会导致ASV IN催化结构域活性位点发生显著的结构修饰。这表明在结构中至少有一个金属结合位点是预先形成的，并表明观察到的酸性残基组合代表一个具有催化活性的活性位点。即使在金属浓度极高的情况下，也仅观察到单个二价阳离子。我们得出结论，要么催化仅需要一个金属离子，要么第二个金属结合位点仅在存在底物和/或蛋白质的其他结构域时才会存在。ASV IN和HIV-1 IN活性位点之间出乎意料的差异仍无法解释；它们可能反映了晶体接触对HIV-1 IN活性位点的影响，或者是结构多态性的一种趋势。
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