Lahm A, Suck D
European Molecular Biology Laboratory, Biological Structures Division, Heidelberg, Germany.
J Mol Biol. 1991 Dec 5;222(3):645-67. doi: 10.1016/0022-2836(91)90502-w.
The structure of a complex between DNase I and d(GCGATCGC)2 has been solved by molecular replacement and refined to an R-factor of 0.174 for all data between 6 and 2 A resolution. The nicked octamer duplexes have lost a dinucleotide from the 3' ends of one strand and are hydrogen-bonded across a 2-fold axis to form a quasi-continuous double helix of 14 base-pairs. DNase I is bound in the minor groove of the B-type DNA duplex forming contacts in and along both sides of the minor groove extending over a total of six base-pairs. As a consequence of binding of DNase I to the DNA-substrate the minor groove opens by about 3 A and the duplex bends towards the major groove by about 20 degrees. Apart from these more global distortions the bound duplex also shows significant deviations in local geometry. A major cause for the observed perturbations in the DNA conformation seems to be the stacking type interaction of a tyrosine ring (Y76) with a deoxyribose. In contrast, the enzyme structure is nearly unchanged compared to free DNase I (0.49 A root-mean-square deviations for main-chain atoms) thus providing a rigid framework to which the DNA substrate has to adapt on binding. These results confirm the hypothesis that groove width and stiffness are major factors determining the global sequence dependence of the enzyme's cutting rates. The nicked octamer present in the crystals did not allow us to draw detailed conclusions about the catalytic mechanism but confirmed the location of the active site near H134 on top of the central beta-sheets. A second cut of the DNA induced by diffusion of Mn2+ into the crystals may suggest the presence of a secondary active site in DNase I.
通过分子置换法解析了脱氧核糖核酸酶I（DNase I）与d(GCGATCGC)2复合物的结构，并对6至2埃分辨率之间的所有数据进行精修，使其R因子达到0.174。带切口的八聚体双链体从一条链的3'端缺失了一个二核苷酸，并通过一个二重轴形成氢键，形成一个14个碱基对的准连续双螺旋。DNase I结合在B型DNA双链体的小沟中，在小沟两侧及其沿线形成接触，总共延伸六个碱基对。由于DNase I与DNA底物结合，小沟开口约3埃，双链体向大沟弯曲约20度。除了这些更全局性的扭曲外，结合的双链体在局部几何结构上也显示出明显偏差。DNA构象中观察到的扰动的一个主要原因似乎是酪氨酸环（Y76）与脱氧核糖之间的堆积型相互作用。相比之下，与游离的DNase I相比，酶的结构几乎没有变化（主链原子的均方根偏差为0.49埃），从而提供了一个刚性框架，DNA底物在结合时必须与之适配。这些结果证实了这样一个假设，即沟宽和刚性是决定酶切割速率全局序列依赖性的主要因素。晶体中存在的带切口的八聚体使我们无法就催化机制得出详细结论，但证实了活性位点位于中央β折叠顶部H134附近。由Mn2+扩散到晶体中诱导的DNA的第二次切割可能表明DNase I中存在一个二级活性位点。
