Formation of a Copper Specific Binding Site in Non-Native States of β-2-Microglobulin

Abstract

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 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 beta2m into amyloid fibers upon binding of Cu(2+). In this work, we demonstrate that while beta2m binds Cu(2+) specifically in the native state, it is binding of Cu(2+) by non-native states of beta2m 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 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: 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, alpha-synuclein, and Abeta, our results may be extended to the emerging field of divalent ion-associated amyloidosis.

Metal binding sheds light on mechanisms of amyloid assembly

“…Zn 2+ and Ni 2+ also bind, but with much weaker affinities. 4,16 Importantly, the binding of Cu 2+ but not Ni 2+ leads to selfassociation. 10 We have previously asserted that at least three modes of Cu 2+ -binding may exist for β2m.…”

“…One is based on coordination to non-native states and is linked to protein destabilization. 16 A second involves Cu 2+ -bound to the monomer in an initial capture complex, and a third represents Cu 2+ bound to oligomeric states. Previous studies have implicated His13, His31, and His51 as well as the N-terminus in native-state metal binding.…”

“…Previous studies have implicated His13, His31, and His51 as well as the N-terminus in native-state metal binding. [16][17][18][19] Of the three histidines above, our own studies have shown that only mutation of His31 affects native-state Cu 2+ affinity. 16 Abbreviations: β2m, β-2 microglobulin; PrP, prion; MHC, major histocompatibility complex; DRA, dialysis-related amyloidosis…”

Unzipping the mysteries of amyloid fiber formation

“…For example, monomeric ␤2m forms amyloid after exposure to divalent Cu(II). However, both the folded and unfolded states of the protein are stabilized as a result of separate binding sites (22). In transthyretin amyloidosis, the soluble form is tetrameric.…”

“…A consequence of the assertion that any protein can form an amyloid (28) is that any subpeptide of any protein can form an amyloid, provided suitable conditions are found. For example, the putative core of IAPP, IAPP [20][21][22][23][24][25][26][27][28][29] , was suggested and then identified as amyloidogenic by using conditions of 10 mg͞ml in 10% acetic acid (29). By contrast, full-length IAPP readily forms fibers at 10 g͞ml.…”