Uncovering the Early Assembly Mechanism for Amyloidogenic β(2)-Microglobulin Using Cross-linking and Native Mass Spectrometry

β(2)-Microglobulin (β(2)m), a key component of the major histocompatibility class I complex, can aggregate into fibrils with severe clinical consequences. As such, investigating the structural aspects of the formation of oligomeric intermediates of β(2)m and their subsequent progression toward fibrillar aggregates is of great importance. However, β(2)m aggregates are challenging targets in structural biology, primarily due to their inherent transient and heterogeneous nature. Here we study the oligomeric distributions and structures of the early intermediates of amyloidogenic β(2)m and its truncated variant ΔN6-β(2)m. We established compact oligomers for both variants by integrating advanced mass spectrometric techniques with available electron microscopy maps and atomic level structures from NMR spectroscopy and x-ray crystallography. Our results revealed a stepwise assembly mechanism by monomer addition and domain swapping for the oligomeric species of ΔN6-β(2)m. The observed structural similarity and common oligomerization pathway between the two variants is likely to enable ΔN6-β(2)m to cross-seed β(2)m fibrillation and allow the formation of mixed fibrils. We further determined the key subunit interactions in ΔN6-β(2)m tetramer, revealing the importance of a domain-swapped hinge region for formation of higher order oligomers. Overall, we deliver new mechanistic insights into β(2)m aggregation, paving the way for future studies on the mechanisms and cause of amyloid fibrillation.