Structural Insights into the Pre-Amyloid Tetramer of β-2-Microglobulin from Covalent Labeling and Mass Spectrometry

Abstract

The main pathogenic process underlying dialysis-related amyloidosis (DRA) is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. This covalent labeling data along with molecular dynamics calculations enable the construction of a tetramer model that indicates how the protein might proceed to form even higher order oligomers.

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

“…More importantly, the binding site that is identified by covalent labeling is consistent with what is known about pre‐amyloid oligomeric β2m structures 21,22 and our recent work on β2m amyloid inhibition by rifamycin SV. 10 Our group recently showed that rifamycin SV inhibits β2m amyloid formation by preventing the β2m dimer from forming an amyloid‐competent tetramer.…”

“…These poses do not agree well with the labeling data. In fact, these docking results position suramin near the known β2m dimer interface, 21,22 which is not consistent with suramin's inability to inhibit β2m amyloid formation or the progression of the oligomers that precede amyloid formation. 10 Thus, we conclude that the docking results are inaccurate, highlighting the value of experimental labeling data.…”

“…10 As indicated above and as previously shown, the β2m tetramer is formed by two dimers interacting between the G β strands and D β strands. 22 Binding of doxycycline to the D β strand, as indicated by our labeling results, reveals how the amyloid formation pathway can be redirected to form non‐amyloid aggregates. It should be noted that Giorgetti et al studied doxycycline/β2m binding by NMR and identified a different binding site that involves β strands A and B.…”

“…We had previously found that the β2m tetramer is formed by two dimers interacting between the G β strands and the D β strands. 22 Considering the binding site identified in the current work, rifamycin SV's proposed mode of action for preventing β2m amyloid formation is to bind near the G β strands (i.e. Figure 2) and prevent two dimers from forming an amyloid‐ competent tetramer.…”

Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues

“…Excess of label reagent can destabilize secondary structures (54). Previous studies have used carbethoxylation by DEPC as a means to probe solvent accessibility (49, 50, 54). Mendoza et al .…”