Chang X, Jorgensen A M, Bardrum P, Led J J
Department of Chemistry, University of Copenhagen, The H. C. Orsted Institute, Universitetsparken 5, DK-2100 Copenhagen O, Denmark.
Biochemistry. 1997 Aug 5;36(31):9409-22. doi: 10.1021/bi9631069.
The three-dimensional solution structure of the phenol-stabilized 36 kDa R6 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(B25) 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 R6 hexamer as the starting model. For both solution hexamers, the average backbone rms deviation is 0.81 A, 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 A for the nonsymmetric and symmetric hexamer, respectively. The overall solution structure of the R6 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.
通过核磁共振光谱法和受限分子动力学确定了苯酚稳定化的36 kDa R6胰岛素六聚体的三维溶液结构。六聚体结构是通过逐步程序推导出来的。最初，通过距离几何方法从665个源自核Overhauser效应（NOE）的距离限制和三个二硫键获得60个单体。随后，使用由最佳的36个单体结构构建的30个六聚体作为起始模型，通过模拟退火计算六聚体结构。核磁共振数据表明，在溶液六聚体中，残基Phe（B25）的芳香环可以采取两种不同的取向：一种是向内指向（分子1，约90%），另一种是从单体表面向外指向（分子2，约10%）。因此，计算了两种六聚体结构：一种由六个分子1单体组成的对称六聚体和一种由五个分子1单体和一个分子2单体组成的非对称六聚体。对于六个单体中的每一个，在六聚体结构计算中使用的限制条件，除了单体内部的限制条件外，还包括胰岛素与苯酚之间的25个NOE、二聚体界面上的23个NOE和两个氢键、三聚体界面上的9个NOE以及分别指定Phe（B25）环不同取向的五个单体内部或两个单体间的NOE。两个锌原子的配位由八个距离限制定义。因此，在两种六聚体计算中分别总共使用了4394和4391个距离限制。在一个迭代过程中，根据NOE限制与单体结构的一致性或不一致性，将其分类为单体内部或单体间的。使用R6六聚体的晶体结构作为起始模型来指定二聚体和三聚体特异性的NOE。对于两种溶液六聚体，如果排除定义不太明确的N端和C端残基，平均主链均方根偏差为0.81 Å。对于非对称和对称六聚体，所有重原子的相应均方根偏差分别为1.17 Å和1.19 Å。R6胰岛素六聚体的整体溶液结构紧凑、刚性且对称，与相应的晶体结构相似。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。
