Solution structures of the R-6 human insulin hexamer
Solution structures of the R-6 human insulin hexamer
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DOI：
10.1021/bi9631069
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发表时间：
1997-08-05
期刊：
影响因子：
2.9
通讯作者：
Led, JJ
中科院分区：
文献类型：
--
作者：
Chang, XQ;Jorgensen, AMM;Led, JJ
采用核磁共振波谱和约束分子动力学方法测定了苯酚稳定的36 kDa R-6胰岛素六聚体的三维溶液结构。六聚体结构的推导采用逐步的程序。最初，通过距离几何，从665个noe导出的距离限制和3个二硫桥中获得了60个单体。随后，以36个最佳单体结构构建的30个六聚体为起始模型，采用模拟退火法计算六聚体结构。核磁共振数据表明，残基苯二酚（B25）的芳香环在溶液六聚体中有两种不同的取向：一种是向内指向（分子1，约90%），另一种是向外指向（分子2，约10%）。因此，我们计算了两种六聚体结构：由6个分子1单体组成的对称六聚体和由5个分子1单体和1个分子2单体组成的非对称六聚体。对于这六种单体，在计算六聚体结构时所使用的约束除单体内约束外，还包括胰岛素和苯酚之间的25个NOEs，二聚体界面上的23个NOEs和两个氢键，三聚体界面上的9个NOEs，以及5个单体内或2个单体间NOEs，分别指定了Phe（R25）环的不同取向。两个Zn原子的配位用8个距离约束来定义。因此，在两个六聚体计算中，分别使用了4394和4391个距离约束。在迭代过程中，NOE约束根据其与单体结构的一致性或不一致性被分类为单体内或单体间。以R-6六聚体的晶体结构为起始模型，对二聚体和三聚体特异性NOEs进行了赋值。对于这两种溶液六聚体，如果排除定义不明确的N端和c端残基，平均主链均方根偏差为0.81埃。对于非对称和对称六聚体，所有重原子对应的均方根偏差分别为1.17和1.19埃。R-6胰岛素六聚体的整体溶液结构紧凑、刚性、对称，类似于相应的晶体结构。然而，表征R态的b链α -螺旋的延伸在溶液结构中比在晶体结构中要短。此外，研究表明，在结构测定的不确定性范围内，Phe（B25）环的取向对分子其余部分的结构没有影响。讨论了这些发现对当前胰岛素受体相互作用模型的重要性。
The three-dimensional solution structure of the phenol-stabilized 36 kDa R-6 insulin hexamer was determined by NMR spectroscopy and restrained molecular dynamics. The hexamer structures were derived using a stepwise procedure. Initially, 60 monomers were obtained by distance geometry from 665 NOE-derived distance restraints and three disulfide bridges. Subsequently, the hexamer structures were calculated by simulated annealing, using 30 hexamers constructed from the best 36 monomer structures as the starting models. The NMR data show that the aromatic ring of residue Phe(B25) can take two different orientations in the solution hexamer: one in which it points inward (molecule 1, about 90%) and one in which it points outward from the surface of the monomer (molecule 2, about 10%). Therefore, two hexamer structures were calculated: a symmetric hexamer consisting of six molecule 1 monomers and a nonsymmetric hexamer consisting of five molecule 1 monomers and one molecule 2 monomer. For each of the six monomers, the restraints used in the calculations of the hexamer structures include, in addition to the intramonomeric restraints, 25 NOEs between insulin and phenol, 23 NOEs and two hydrogen bonds across the dimer interface, nine NOEs across the trimer interface, and five intramonomeric or two intermonomeric NOEs, respectively, specifying the different orientations of the Phe(R25) ring. The coordination of the two Zn atoms was defined by eight distance restraints. Thus, a total of 4394 and 4391 distance restraints, respectively, were used in the two hexamer calculations. The NOE restraints were classified in an iterative process as intra- or intermonomeric on the basis of their consistency or inconsistency with the structure of the monomer. The assignment of the dimer- and trimer-specific NOEs was made using the crystal structure of the R-6 hexamer as the starting model. For both solution hexamers, the average backbone rms deviation is 0.81 Angstrom, if the less well-defined N- and C-terminal residues are excluded. The corresponding rms deviations for all heavy atoms are 1.17 and 1.19 Angstrom for the nonsymmetric and symmetric hexamer, respectively. The overall solution structure of the R-6 insulin hexamer is compact, rigid, and symmetric and resembles the corresponding crystal structure. However, the extension of the B-chain alpha-helix, which characterizes the R state, is shorter in the solution structure than in the crystal structure. Also, the study shows that the orientation of the Phe(B25) ring has no effect on the structure of the rest of the molecule, within the uncertainty of the structure determination. The importance of these findings for the current model for the insulin-receptor interaction is discussed.