Putnam C D, Shroyer M J, Lundquist A J, Mol C D, Arvai A S, Mosbaugh D W, Tainer J A
Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
J Mol Biol. 1999 Mar 26;287(2):331-46. doi: 10.1006/jmbi.1999.2605.
Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi). A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity. High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi. The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes. Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA. Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex. Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands. A similar beta-zipper is observed in the conversion between the open and closed forms of UDG. The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity.
尿嘧啶-DNA糖基化酶（UDG）是DNA碱基切除修复中的一种关键酶，可识别并从DNA中去除尿嘧啶，它会被热稳定的尿嘧啶-DNA糖基化酶抑制蛋白（Ugi）特异性且不可逆地抑制。Ugi对UDG的高度特异性抑制存在一个矛盾之处，即Ugi如何能成功模拟UDG与DNA主链的相互作用，而又不会与众多具有DNA主链结合亲和力的其他酶产生显著的交叉反应。Ugi游离状态以及与野生型和功能缺陷型His187Asp突变体大肠杆菌UDG形成复合物的高分辨率X射线晶体结构揭示了Ugi模拟双链DNA主链的详细分子基础。Ugi的整体形状和电荷分布与B型DNA和UDG:DNA产物复合物中观察到的扭结DNA之间轨迹的中点最为相似。因此，Ugi靶向UDG的尿嘧啶翻转机制，似乎是UDG将尿嘧啶核苷酸从DNA中翻转的过渡态模拟物。基本上，高亲和力UDG-Ugi相互作用所需的所有精确形状、静电和疏水互补性都是预先存在的，除了Ugi的Gln19羰基和Glu20侧链的一个关键翻转，它是由复合物的形成触发的。未结合的Ugi与与UDG复合的Ugi之间的构象变化涉及β-拉链结构基序，我们因其在分子内β-链之间观察到的可逆配对而命名。在UDG的开放和封闭形式的转换中也观察到类似的β-拉链。Ugi中极高水平的预先存在的与UDG特异性DNA结合特征的结构互补性与关键局部构象变化的结合解决了UDG-Ugi的矛盾，并为形成避免交叉反应的非常高亲和力的DNA酶-抑制剂复合物提出了一种潜在的通用结构解决方案。
