Copper Binding to β-2-Microglobulin and Its Pre-Amyloid Oligomers

Abstract

Abstractβ-2-microglobulin (β2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes β2m to organize into native-like dimers and tetramers that precede amyloid formation. Cu(II) is then released from higher order oligomers before mature Cu(II)-free amyloid fibrils are formed. While some of the Cu(II)-induced structural changes that enable β2m self assembly are starting to be revealed, the details of how the Cu(II) binding site evolves from the monomer to the dimers and tetramers are not known. Here, we report results from three mass spectrometry (MS) based methods that provide insight into the changing Cu-β2m interactions. We find that monomeric β2m binds Cu(II) via the N-terminal amine, the amide of Gln2, His31, and Asp59. In the dimer and tetramer, Asp59 is no longer bound to Cu(II), but the other residues still comprise a well-defined albeit weaker binding site that is better able to release Cu(II). Consistent with this is the observation that a fraction of the tetrameric species no longer binds Cu(II) at this weakened binding site, which agrees with a previous report that suggested the tetramer as the first Cu(II)-free oligomer. Our results also provide some insight into structural changes caused by Cu(II) binding that facilitate oligomer formation. Specifically, binding by Asp59 in the monomer requires significant movement of this residue, and we propose that this repositioning is important for establishing a pair of dimer-stabilizing salt bridges between this residue and Lys19. We also find evidence that Cu(II) binding in the Nterminal region of the monomer repels Arg3, which likely allows this residue to form a pair of dimerstabilizing salt bridges with Glu16. Overall, our measurements suggest that the previously proposed conformational switch caused by Cu(II) binding includes not only a cis-trans isomerization at Pro32 but also the repositioning of residues that are critical for the formation of new electrostatic interactions.β-2-microglobulin (β2m) is a 12 kDa subunit of the class I major histocompatibility complex and is a structural unit essential for the cell-surface expression of this complex. During normal turnover, β2m is released into serum and is eventually catabolized by the kidney. In patients undergoing hemodialysis as a result of kidney failure, β2m concentrations become elevated in the serum, and after as little as 18 months, β2m amyloids begin to form in the joints of these patients [1]. The cause of β2m amyloid formation in vivo is not precisely known, but several † This material is based upon work supported by the National Institutes of Health Grant RO1 GM 075092 *Department of Chemistry, University of Massachusetts, Amherst, rwvachet@chem.umass.edu, Telephone: (413) 545-2733, Fax: (413) 545-4490. ‡ Current address: Department of Chemistry, U-3060, University of Connecticut, 55 North Eagleville Road, Storrs, CT 06269-3060Supporting Information Ava...

DE‐loop mutations affect β2 microglobulin stability, oligomerization, and the low‐pH unfolded form

“…The effect of the D59P mutation can be interpreted according to previously published evidence on electrostatic interactions. A salt bridge between Asp59 and Lys19 has been observed at the subunit interface of hexameric b2m 16,28,29 and has been implicated in dimer association. 28,29 The effect of Cu 2þ on protein oligomerization has also been interpreted by repositioning of Asp59 into a more favorable orientation for ion pairing with Lys19.…”

“…A salt bridge between Asp59 and Lys19 has been observed at the subunit interface of hexameric b2m 16,28,29 and has been implicated in dimer association. 28,29 The effect of Cu 2þ on protein oligomerization has also been interpreted by repositioning of Asp59 into a more favorable orientation for ion pairing with Lys19. 28,29 Our observation that the D59P mutant has a reduced propensity to native aggregation is consistent with a contribution of Asp59 in w.t.…”

“…28,29 The effect of Cu 2þ on protein oligomerization has also been interpreted by repositioning of Asp59 into a more favorable orientation for ion pairing with Lys19. 28,29 Our observation that the D59P mutant has a reduced propensity to native aggregation is consistent with a contribution of Asp59 in w.t. protein association even in the absence of Cu 2þ .…”

Micronutrient Metabolism in Hemodialysis Patients

“…Elevations in plasma Cu can increase protein kinase C activation due to oxidative stress and these are associated with inflammation and the progression of renal diseases (Davis et al, 2001). Recently, β-2-microglobulin deposits in the form of amyloid fibrils were found in the musculoskeletal systems of dialysis patients as a result of kidney failure (Srikanth et al, 2009). β-2-microglobulin accumulation with resultant tumor formation is also a known, albeit rare complication of long-term hemodialysis (Mendoza et al, 2010).…”

“…β-2-microglobulin has been reported to be markedly correlated with oxidative stress and inflammatory biomarkers in hemodialysis patients (Filiopoulos et al, 2009). However, Cu binding to β-2-microglobulin may precede amyloid formation (Srikanth et al, 2009). Elevated serum and leukocyte Cu concentrations were associated with cardiovascular disease; Cu may directly affect atherogenesis and is a marker of inflammation associated with atherosclerosis (Kinsman et al, 1990;Ford, 2000).…”