Crystallographic refinement and structure of DNase I at 2 A resolution.
Crystallographic refinement and structure of DNase I at 2 A resolution.
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DOI：
10.1016/0022-2836(86)90280-9
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发表时间：
1986-12
影响因子：
5.6
通讯作者：
C. Oefner;D. Suck
中科院分区：
文献类型：
--
作者：
C. Oefner;D. Suck
用Hendrickson和Konnert的约束参数倒数最小二乘法对牛胰腺脱氧核糖核酸酶I(DNase I)的结构进行了2？分辨率的精炼。I≥3σ(I)分辨率为6.0时，16,104次反射的传统R因子为0.157。改进后的结构的键长和键角与理想的均方根值接近。偏差分别为0.023°和1.4°。火箭队。短的非粘合触点与范德华半径之和的偏差为0.18？侧链的取向显示出与交错构象对应的χ1角在−60°、180°、60°(按优先顺序)的明显三峰分布。化学确定的序列在四个位置被校正，主要的校正是在Arg27和Arg28之间插入三肽Ile-Val-Arg。在两个中心六链β折叠薄片之间和两侧扩展的疏水区域是2033年蛋白质原子平均各向同性温度因子11.9ä2的主要原因。除了Gly97和Gly102之间的柔性环区(Glu99和Ser100无序)和碳水化合物侧链都延伸到一个大的溶剂通道外，只有暴露的环Arg70到Lys74表现出较高的热迁移率。在DNaseI的8个螺旋中最长的一个，总共占结构的26%，有一个22°的扭结，由两个α-螺旋片段(第136-144和145-155残基)组成，由310个螺旋旋转隔开。DNase I片段1至120和121至257可由大约2倍轴(R.M.S.61个相同的Cα位的偏差为1.49°)，表明该酶可能是基因复制的结果。在结晶条件下与DNase I结合的两个钙离子对其结构完整性很重要，因为它们稳定了表面环Asp198到Thr204，并将柔性环中的高热迁移率区域限制在Gly97到Gly102残基。连接在ASN18上的N-连接碳水化合物侧链是高甘露糖型的，分支点在第3位的甘露糖残基上。与蛋白质接触的两个N-乙酰氨基葡萄糖残基和随后的甘露糖被很好地定义，并在延伸的构象中显示出通常的β-1，4-键，而另外四个甘露糖残基的位置由于越来越高的热运动和可能的微观异质性而不太可靠。在差分傅里叶图中确定了377个结晶上独立的溶剂位，并对它们的位置和温度因子进行了修正(=30Å2)。讨论了一种可能的作用机制和与双链DNA相互作用的模型，该模型涉及到小槽中暴露的环(Arg70到Lys74)的结合。
The structure of bovine pancreatic deoxyribonuclease I (DNase I) has been refined at 2 Å resolution using the restrained parameter, reciprocal least-squares procedure of Hendrickson and Konnert. The conventional R-factor for 16,104 reflections with I ≥ 3σ(I) from 6.0 to 2.0 Å resolution is 0.157. Bond lengths and angles of the refined structure are close to ideal values with root-mean-square (r.m.s.) deviations of 0.023 Å and 1.4 °, respectively. The r.m.s. deviation of short non-bonded contacts from the sum of van der Waals' radii is 0.18 Å. The orientation of side-chains shows a clear trimodal distribution of χ1-angles at −60 °, 180 °, 60 ° (in the order of preference) corresponding to staggered conformations. The chemically determined sequence was corrected at four positions, the major correction being an insertion of the tripeptide Ile-Val-Arg between Arg27 and Arg28. Extended hydrophobic regions in between, and on either side of, the two central six-stranded β-pleated sheets are mainly responsible for the low average isotropic temperature factor of 11.9 Å2 for the 2033 protein atoms. Besides the flexible loop region between Gly97 and Gly102 (Glu99 and Ser100 are disordered) and the carbohydrate side-chain, which both extend into a large solvent channel, only the exposed loop Arg70 to Lys74 shows elevated thermal mobility. The longest of the eight helices in DNase I, together representing 26% of the structure, has a 22 ° kink and consists of two α-helical segments (residues 136 to 144 and 145 to 155) separated by a 310-helical turn. DNase I fragments 1 to 120 and 121 to 257 can be superimposed by an approximate 2-fold axis (r.m.s. deviation 1.49 Å for 61 equivalent Cα positions), suggesting that the enzyme might be the result of gene duplication. The two Ca2+ bound to DNase I under crystallization conditions are important for its structural integrity by stabilizing the surface loop Asp198 to Thr204 and limiting the region of high thermal mobility in the flexible loop to residues Gly97 to Gly102. The N-linked carbohydrate side-chain attached to Asn 18 is of the high-mannose type with a branching point at the mannose residue in position 3. Two N-acetylglucosamine residues, which are in contact with the protein, and the following mannose are well defined and show the usual β-1,4-linkage in an extended conformation, whereas the positions of four further mannose residues are less reliable due to increasingly higher thermal motion and possibly microheterogeneity. A total of 377 crystallographically independent solvent sites has been determined in difference Fourier maps and their positions and temperature factors have been refined ( = 30 Å2). A possible mechanism of action and a model for the interaction with double-stranded DNA involving the binding of an exposed loop (Arg70 to Lys74) in the minor groove is discussed.