A regulatable switch mediates self-association in an immunoglobulin fold

Abstract

β-2 microglobulin (β2m) is a globular protein that self-associates into fibrillar amyloid deposits in patients undergoing hemodialysis therapy. Formation of these β-sheet–rich assemblies is a fundamental property of polypeptides that can be triggered by diverse conditions. For β2m, oligomerization into pre-amyloidogenic states occurs in specific response to coordination by Cu2+. Here we report the basis for this self-association at atomic resolution. Metal is not a direct participant in the molecular interface. Rather, binding results in distal alterations enabling the formation of two new surfaces. These interact to form a closed hexameric species. The origins of this include isomerization of a buried and conserved cis-proline previously implicated in the β2m aggregation pathway. The consequences of this isomerization are evident and reveal a molecular basis for the conversion of this robust monomeric protein into an amyloid-competent state.

Metal binding sheds light on mechanisms of amyloid assembly

“…Within each hexamer, six Cu 2+ atoms are bound consistent with the binding stoichiometry observed in solution. 23,25 However, contrary to our initial expectation, 10 metal does not localize to interfaces and does not bridge adjacent subunits. Rather, Cu 2+ binding is entirely intramolecular where all coordinating ligands are derived from a single polypeptide chain.…”

“…Cu 2+ binding to β2m and PrP. (A) Cu 2+ -site derived from the Cu 2+ -bound β2m hexamer (PDB 3CIQ) 25 and (B) Cu 2+ -site derived from the HGGGW fragment of the PrP octapeptide repeat. 26 Both sites display coordination by an imidazole ring, two backbone nitrogens and a backbone carbonyl in a square-planar arrangement.…”

“…The structure we have recently reported describes the Cu 2+ binding site within the context of an oligomer. 25 Formation of this site requires disruption of the first β-strand and local rearrangements proximal to His31. This includes rotation of Phe30 from the hydrophobic core to face solvent, and the cis-trans isomerization of Pro32.…”

“…Nevertheless, the global stability, Cu 2+ affinity, and rate of oligomerization remain similar for H13F and WT β2m. 16,25 The hexamer is organized as a closed ring surrounding a solvent-filled central channel. The ring is three-fold symmetric demonstrating the existence of two distinct classes of interface ( Figure 1).…”

NMR reveals novel mechanisms of protein activity regulation

“…Proline cis-trans isomerization has emerged as a particularly efficient regulatory mechanism in many biological processes, including cell signaling, [62][63][64][65] neurodegeneration, 66 amyloidogenesis, 67 channel gating, 68 gene regulation, 69,70 phage and virus infection, 71,72 enzyme function, 73,74 and ligand recognition. 75 Proline isomerization is unique in that it exerts its function through multiple mechanisms involving (i) significant conformational changes caused by the 180 rotation about the prolyl bond, (ii) slow kinetics of isomerization affording a molecular timer, and (iii) the recruitment of prolyl cis-trans isomerase enzymes (PPIases).…”