Formation of a copper specific binding site in non-native states of β-2-microglobulin
Formation of a copper specific binding site in non-native states of β-2-microglobulin
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DOI：
10.1021/bi025944a
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发表时间：
2002-08-27
期刊：
影响因子：
2.9
通讯作者：
Miranker, AD
中科院分区：
文献类型：
--
作者：
Eakin, CM;Knight, JD;Miranker, AD
长期血液透析的一个使人衰弱的并发症是β 2-微球蛋白（β 2 m）在关节间隙中以淀粉样斑块的形式沉积。我们最近发现，在透析治疗期间遇到的浓度下，Cull可能是一个促成因素（如果不是因果关系）。这种效应的基础是Cull结合后β 2 m的不稳定性和掺入淀粉样纤维。在这项工作中，我们证明，虽然β 2 m结合铜+特异性在自然状态下，它是由β 2 m的非自然状态的铜2+的结合，这是负责不稳定。潜在的Cu 2+的配位基团的诱变表明，天然状态的Cu 2+的结合是介导的残基和结构，是不同于那些结合在非天然状态。与原生状态相比，非原生状态对铜的亲和力增加导致整体不稳定。使用质谱，核磁共振和荧光技术，我们表明，本地化的自然状态结合H31和W 60，是高度特异性的Cu 2+超过Zn 2+和Ni 2+。在β 2 m的非天然状态下，Cu 2+的结合由残基H13、H51和H84介导，但不是H31。虽然变性的β 2 m具有整体未折叠状态的特征，但它仍然表现出以下强的结合特异性：Cu 2 + > Zn 2 + >> Ni 2+。这需要在该蛋白质的未折叠状态下存在明确定义的结构。由于在许多其他淀粉样变性中报道了Cu 2+效应，例如，PrP，α-突触核蛋白和Abeta，我们的研究结果可能会扩展到二价离子相关淀粉样变性的新兴领域。
A debilitating complication of long-term hemodialysis is the deposition of beta-2-microglobulin (beta2m) as amyloid plaques in the joint space. We have recently shown that Cull can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of beta2m into amyloid fibers upon binding of Cull. In this work, we demonstrate that while beta2m binds Cu2+ specifically in the native state, it is binding of Cu2+ by nonnative states of beta2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu2+ shows that native state binding of Cu2+ is mediated by residues and structures that are different than those which bind in non-native states. An increased affinity for copper by non-native states compared to that of the native state gives rise to overall destabilization. Using mass spectrometry, NMR, and fluorescence techniques, we show that native state binding is localized to H31 and W60 and is highly specific for Cu2+ over Zn2+ and Ni2+. Binding of Cu2+ in non-native states of beta2m is mediated by residues H13, H51, and H84, but not H31. Although denatured beta2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu2+ > Zn2+ >> Ni2+. This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu2+ effects are reported in many other amyloidoses, e.g., PrP, alpha-synuclein, and Abeta, our results may be extended to the emerging field of divalent ion-associated amyloidosis.