Formation of a Copper Specific Binding Site in Non-Native States of β-2-Microglobulin†
A debilitating complication of long-term hemodialysis is the deposition of -2-microglobulin ( 2m) as amyloid plaques in the joint space. We have recently shown that Cu 2+ can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of 2m into amyloid fibers upon binding of Cu 2+ . In this work, we demonstrate that while 2m binds Cu 2+ specifically in the native state, it is binding of Cu 2+ by
... onnative states of 2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu 2+ shows that native state binding of Cu 2+ 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 Cu 2+ over Zn 2+ and Ni 2+ . Binding of Cu 2+ in non-native states of 2m is mediated by residues H13, H51, and H84, but not H31. Although denatured 2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu 2+ > Zn 2+ . Ni 2+ . This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu 2+ effects are reported in many other amyloidoses, e.g., PrP, R-synuclein, and A , our results may be extended to the emerging field of divalent ion-associated amyloidosis. 1 Abbreviations: 2m, -2-microglobulin; CD, circular dichroism spectroscopy; DRA, dialysis-related amyloidosis; ESI-MS, electrospray ionization mass spectrometry; MHC, major histocompatibility complex; NMR, nuclear magnetic resonance spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; rWT, wild-type human 2m from E. coli, possessing an additional N-terminal methionine; hWT, human-derived 2m.