Light chain amyloidosis (AL) is a protein misfolding disorder caused by the abnormal proliferation of clonal plasma B cells that excessively secrete monoclonal antibody light chains into the bloodstream; these proteins deposit as amyloid fibers in organs and tissues [[1], [2], [3]]. All organs can be affected by these structures, even the central nervous system in very rare cases, leading to irreversible organ dysfunction and eventual patient death if not promptly diagnosed or effectively treated [4,5].

AL, also known as primary amyloidosis, is a rare disease. A recent study found a worldwide prevalence rate of ∼50 cases per million people over 20 years and a crude annual incidence rate of ∼10 cases per million people, generally affecting the population between 50 and 80 years of age [[6], [7], [8]]. The average life expectancy of patients with AL after diagnosis is approximately 3 years, although it may be shortened to 6 months in patients with cardiac damage. AL is the most common systemic amyloidosis, and the clinical manifestations depend on the affected organ. However, common symptoms have been defined to facilitate the diagnosis: macroglossia, periorbital purpura, inflammation of the submandibular gland, skin hemorrhages, blood pressure changes, and excess antibody light chains in urine and blood [9].

Several studies have shown the close relationship between AL and the antibody light chain λVI (6a) family proteins. The prevalence of 6a family proteins can reach up to 30 % in the disease, whereas they are expressed by only 2 % of plasma B cells in healthy individuals [[10], [11], [12]]. An allelic variant within family 6a was found to have the R24G mutation; this allele is found in 25 % of the proteins in this group. Notably, the three-dimensional structure by Cryo-EM of an antibody light chain amyloid fiber from the heart and kidneys of an AL patient with severe amyloid cardiomyopathy and kidney damage presents this R24G mutation as a background to the changes associated with antibody maturation [[13], [14], [15], [16], [17]].

In vitro studies with two recombinant proteins, one containing the germline-encoded 6a sequence and the other containing the R24G mutation fused with the frequently expressed J segment JL2, 6aJL2 and 6aJL2-R24G respectively, showed that the R24G variant is thermodynamically less stable and forms amyloid fibers more efficiently, due not only to the loss of interactions caused by the mutation but also to changes in the dynamics of the tertiary structure [14,18,19]. The 3D structure of 6aJL2-R24G is shown in Fig. 1.

Several factors may promote structural perturbations leading to aggregation of antibody light chains into amyloid fibers, including oxidative stress [20], mutations [21], and interactions with glycosaminoglycans [22]. The role of metal ions, particularly Cu(II) in protein toxicity and aggregation, both in vivo and in vitro, has been extensively studied in different systems. Although there are few reports regarding the effect of metal ions on the development of AL, studies have demonstrated the binding ability of immunoglobulins to Cu(II) both in vivo and in vitro [[23], [24], [25], [26], [27], [28]].

Previously, we reported for the first time the interaction of Cu(II) with 6aJL2-R24G [29]. We found that the protein has two Cu(II) binding sites in its native state with submicromolar affinities. One of the proposed binding sites involves His99, Asp95, and Asn1, and three water molecules; the second site involves His8, Ser9, and three water molecules. According to our molecular dynamics simulations, Cu(II) binding only to His99 induces the loss of interactions between the CDR3 and CDR1, and the loop C-C′ and CDR1, which promotes the fibril formation. Meanwhile, Cu(II) binding only to His8 results in few changes in the protein dynamics. However, when Cu(II) is bound to both histidines, the destabilizing effect is stronger, and the dynamics and intramolecular interactions of the protein ultimately promote the formation of amyloid fibers [29]. In this work, we evaluated the role of His99 and His8 on Cu(II) binding by mutagenesis, fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry. The promotion of amyloid fibers was determined by transmission electron microscopy. His99 was deleted as this residue is lost in the 77 % of the λ light chains reported in AL-Base [30]. On the other hand, His8 was substituted by Ser given that this substitution had been previously studied in the context of the 6aJL2 protein [31]. Molecular dynamics simulations of 6aJL2-R24G and the mutants in the absence of Cu(II) were used to explain the molecular changes induced by the mutations; the simulations of 6aJL2-R24G with Cu(II) are already published [29].