Delineating the Conformational Elements Responsible for Cu2+-Induced Oligomerization of β-2 Microglobulin

beta-2 microglobulin (beta 2m) Is a small globular protein implicated in amylold fiber formation in renal patients on long-term hemodialysis therapy. In vitro, under physiological conditions, beta 2m is not aggregation prone. However, in the presence of stoichiometric Cu2+, beta 2m readily self-associates ultimately leading to heterogeneously sized aggregates. As this process occurs under near physiological solution conditions where the fold is >= 20 kJ/mol stabilized over the unfolded state, local conformational rearrangements are critical to understanding the oligomerization of beta 2m. The isomerization of a conserved cis proline at residue 32 is a recognized step in this process that can be initiated by Cu2+ binding. To better understand the structural basis of metal-induced oligomerization of beta 2m, we set out to determine the role of individual imidazole side chains in mediating metal binding affinity, native state stability, and oligomerization in the framework of P32A beta 2m. We find that P32A in the presence of Cu2+ forms a tetramer in an apparently cooperative manner. One interface of this tetramer appears to reside along an edge strand as H51 is a key residue in mediating oligomerization. Furthermore, H31 is the main Cu2+ binding residue in P32A and has an important role in stabilizing the protein in its holo form. Importantly, Cu2+ binding affinity in P32A is much greater than in WT. Here, We show that this strong binding affinity need not be directly coupled to oligomerization. We interpret Our results in terms of the known structures of beta 2m(apo) and a reversible hexameric state of beta 2m(holo).