胰岛素的 1H 傅里叶变换核磁共振研究：Ca2+与 Glu(B13)位点的配位驱动六聚体组装并诱导构象变化。

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change.

作者信息

Palmieri R, Lee R W, Dunn M F

机构信息

Department of Biochemistry, University of California, Riverside 92521.

出版信息

Biochemistry. 1988 May 3;27(9):3387-97. doi: 10.1021/bi00409a040.

DOI:10.1021/bi00409a040

PMID:2898949

Abstract

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.

摘要

在(^{2}H_{2}O)中，以pH为函数进行了金属离子与胰岛素结合的(^{1}H)傅里叶变换核磁共振研究，以确定金属离子与Glu(B13)位点配位对胰岛素六聚体组装和结构的影响。已对分别含有一个(Ca^{2 +})和两个(Zn^{2 +}/)六聚体以及三个(Cd^{2 +}/)六聚体的胰岛素物种的(^{1}H)核磁共振谱的C - 2组氨酸区域进行了归属。(Cd^{2 +})衍生物((In){6}(Cd^{2 +}){2}Cd^{2 +})（其中两个(Cd^{2 +})离子与His(B10)位点配位，其余(Cd^{2 +})离子与Glu(B13)位点配位[Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560]）以及(Zn^{2 +}-Ca^{2 +})衍生物((In){6}-(Zn^{2 +}){2}Ca^{2 +})（其中两个(Zn^{2 +})离子与His(B10)位点配位，(Ca^{2 +})离子与Glu(B13)位点配位）给出的谱图中，His(B10)的C - 2质子共振相对于无金属胰岛素向高场移动。发现在pH为8.6至10的区域中，含有In: (Zn^{2 +}) = 6:2比例的胰岛素溶液（3 - 20 mg/mL）的谱图中既包含无金属胰岛素物种的信号，也包含(2Zn -)胰岛素六聚体((In){6}(Zn^{2 +}){2})的信号。发现添加(Ca^{2 +})（比例为In: (Zn^{2 +}): (Ca^{2 +}) = 6:2:1）或40 mM NaSCN可提供足够的额外热力学驱动力，使胰岛素六聚体几乎完全组装。In: (Cd^{2 +}) = 6:3比例的(Cd^{2 +})也能驱动六聚体组装完成。我们推测(Ca^{2 +})和(Cd^{2 +})提供的额外热力学驱动力是由于这些金属离子与六聚体的Glu(B13)羧酸盐配位。在高pH*下，这种配位中和了六个Glu(B13)羧酸盐之间的排斥性库仑相互作用，并在二聚体 - 二聚体界面形成金属离子“交联”。对((In){6}(Zn^{2 +}){2})与((In){6}(Zn^{2 +}){2}Ca^{2 +})、((In){6}(Cd^{2 +}){2}Cd^{2 +})和((In){6}(Cd^{2 +}){2}Ca^{2 +})的(^{1}H)核磁共振谱的芳香区域进行比较表明，(Ca^{2 +})或(Cd^{2 +})与Glu(B13)位点的结合会诱导构象变化，该变化扰乱了胰岛素结构中几个芳香族残基侧链的环境。由于这些残基位于单体 - 单体和二聚体 - 二聚体亚基界面上，我们得出结论，构象变化包括亚基界面的微小变化，这些变化改变了芳香环的微环境。