Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G

Abstract

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.

Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains

“…While the C L domain is destabilized in the H7-H188A/H197A mutant, the V L domain is stabilized by its interaction with copper. The observed copper-induced stabilization of H7-V L is in contrast to a previous study, in which copper binding destabilized the V L domain of the LC 6αJL2-R24G, thus accelerating fibril formation [47]. We noted that the unique sequence of each individual LC V L -domain generated distinct molecular features that could lead to the observed (de-)stabilization differences caused by Cu binding.…”

Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains

“…38,39 The equilibrium and kinetic properties of unfolding as well as the in vitro fibril formation of these two proteins have been studied under different experimental conditions using chaotropic agents (urea and GdnCl), metal ions, and changes in temperature or pH. 12,32,33,35,40,41 In addition, the dynamics of 6aJL2 has been studied by NMR and computer simulations, 37,42 where it was found that strands B, C, and E−G were the most stable. 42 Studies at low pH have shown that the EF loop, which connects strands E and F, undergoes a conformational rearrangement that destabilizes the protein at acidic pH.…”

Unveiling the Effects of Copper Ions in the Aggregation of Amyloidogenic Proteins

“…This suggests that the formation of aggregates involves the participation of partially unfolded intermediaries as supported by a recent paper [ 57 ]. Cu 2+ has been associated with the development of degenerative diseases, showing affinities similar to those observed for 6aJL2-R24G [ 59 ]. Therefore, the binding of Cu 2+ to 6aJL2-R24G could potentially trigger the aggregation under physiological conditions.…”