Osborne M J, Breeze A L, Lian L Y, Reilly A, James R, Kleanthous C, Moore G R
School of Biological Science, University of East Anglia, Norwich, U.K.
Biochemistry. 1996 Jul 23;35(29):9505-12. doi: 10.1021/bi960401k.
The 86-amino acid colicin E9 immunity protein (Im9), which inhibits the DNase activity of colicin E9, has been overexpressed in Escherichia coli and isotopically enriched with 15N and 13C. Using the 3D CBCANH and CBCA(CO)NH experiments, we have almost completely assigned the backbone 13C resonances and extended previously reported 15N/1H backbone assignments [Osborne et al. (1994), Biochemistry 33, 12347-12355]. Side chain assignments for almost all residues were made using the 3D 13C HCCH-TOCSY experiment allied to previous 1H assignments. Sixty solution structures of Im9 were determined using the DIANA program on the basis of 1210 distance restraints and 56 dihedral angle restraints. The 30 lowest-energy structures were then subjected to a slow-cooling simulated annealing protocol using XPLOR and the 21 lowest-energy structures, satisfying the geometric restraints chosen for further analysis. The Im9 structure is well-defined except for the termini and two solvent-exposed loops between residues 28-32 and 57-64. The average RMSD about the average structure of residues 4-84 was 0.94 A for all heavy atoms and 0.53 A for backbone C alpha, C = O, and N atoms. The Im9 fold is novel and can be considered a distorted antiparallel four-helix bundle, in which the third helix is rather short, being terminated close to its N-terminal end by a proline at its C-terminus. The structure fits in well with available kinetic and biochemical data concerning the interaction between Im9 and its target DNase. Important residues of Im9 that govern specificity are located on the molecular surface in a region rich in negatively charged groups, consistent with the proposed electrostatically steered association [Wallis et al. (1995a), Biochemistry 34, 13743-13750].
86个氨基酸的大肠杆菌素E9免疫蛋白（Im9）可抑制大肠杆菌素E9的DNase活性，该蛋白已在大肠杆菌中过表达，并通过15N和13C进行了同位素富集。利用3D CBCANH和CBCA(CO)NH实验，我们几乎完全确定了主链13C共振，并扩展了先前报道的15N/1H主链归属[奥斯本等人（1994年），《生物化学》33卷，12347 - 12355页]。几乎所有残基的侧链归属都是使用与先前1H归属相关联的3D 13C HCCH - TOCSY实验完成的。基于1210个距离约束和56个二面角约束，使用DIANA程序确定了Im9的60个溶液结构。然后使用XPLOR对30个最低能量结构进行慢冷模拟退火方案处理，得到21个最低能量结构，满足为进一步分析所选的几何约束。除了末端以及残基28 - 32和57 - 64之间的两个溶剂暴露环外，Im9结构定义明确。对于残基4 - 84的平均结构，所有重原子的平均均方根偏差（RMSD）为0.94 Å，主链Cα、C = O和N原子的平均均方根偏差为0.53 Å。Im9的折叠结构是新颖的，可被视为一种扭曲的反平行四螺旋束，其中第三个螺旋相当短，在其C末端被一个脯氨酸靠近其N末端终止。该结构与关于Im9与其靶标DNase相互作用的现有动力学和生化数据非常吻合。决定特异性的Im9重要残基位于分子表面富含带负电荷基团的区域，这与所提出的静电引导结合一致[沃利斯等人（《生物化学》34卷，13743 - 13750页，1995年a）]。
