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

Abstract

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu²⁺ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu²⁺-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Keywords: copper binding; copper ions; light chain amyloidosis; protein aggregation; soluble toxicity.

Figure 1

Combined MST, ITC and thermal unfolding demonstrate low micromolar binding affinity of copper to the V_L domain of H7. (A) The crystal structure of H7 (PDB: 5MUH) exemplifies the typical two-domain architecture and dimer interface observed for most LC structures [28]. Both the V_L and C_L immunoglobulin domains contribute to the dimer interface. The strands, helices and loops of one monomer are shown in red, yellow and grey. The second subunit is shown in grey. The positions of H197 and H188 are highlighted. (B) The color legend used throughout the manuscript unless stated otherwise. H7, H7-H188A/H197A and M7 were colored in tones of red, blue and black, respectively. (C,D) Selected MST time-traces and the baseline-corrected ITC thermograms are shown in the panels below the final MST titration curves and ITC binding isotherms. The complete set of MST and ITC raw data is shown in Figures S1 and S2. MST time-traces are color-coded on a log2-concentration scale. (C) The MST binding curve averaged from four Cu²⁺-to-H7 titration experiments (red) reveals a sigmoidal line-shape with an ΔF_norm amplitude of 15‰ and a fitted K_D value of 750 ± 60 nM. The titration curves of Ca²⁺, Fe²⁺, Mg²⁺, Na⁺ and Zn²⁺ to H7 over similar concentration ranges are summarized in the “ions” panel (light orange). The associated MST-TT of a single Mg²⁺ titration is shown below, all other raw data are shown in Figure S1. The titration of Cu²⁺ to M7-wt (black) yielded a binding curve with an ΔF_norm amplitude of 15‰ and a fitted K_D value 50 ± 15 μM, derived from two experiments. At the highest Cu²⁺ concentrations, we noticed wavy MST time-traces indicative for Cu²⁺ induced M7 aggregation, which were not included in the final analysis. (D) Offset-corrected ITC-binding isotherms demonstrated sub-micromolar affinity for the binding of Cu²⁺ to H7 (red) with K_D and binding the enthalpy (ΔH) values of 900 ± 100 nM and −8.7 ±  0.1 kcal/mole, respectively, as derived from global fits to four experiments. The substitution of His-188 and H-197 to Ala residues did not significantly alter the binding, yielding fitted K_D and ΔH values of 900 ±  300 nM and −7.6 ±  0.8 kcal/mole (blue). Chelation of Cu²⁺ by molar excess of EDTA abolished binding (dark red). The binding isotherm derived from two experiments for binding of Cu²⁺ to M7 (black) was fit with K_D and enthalpy (ΔH) values of 16 ± 6 μM and 6 ±  1 kcal/mole. (E) Thermal unfolding monitored at a 350/330 nm fluorescence ratio in nanoDSF revealed two melting temperatures (T_m) for the unfolding of H7. The binding of copper (dark red) shifted the first Tm value from 45 to 48.3 °C. The thermal melting of H7 in the presence of EDTA (pink curve) yielded a profile almost identical to H7 in the absence of copper (light red).

Figure 2

Mutated H7 has a similar overall structure but destabilized C_L domain compared to WT, and its capacity to produce H₂O₂ is unaltered. (A) CD spectra recorded for H7 (red) and H7-H188A/H197A (light blue) indicated minimal differences below 210 nm that are possibly caused by minimal loss of the strand and/or helix secondary structure in the C_L domain. (B) H7 and H7-H188A/H197A eluted from the analytical SEC column Superdex 200 10/300 GL at almost identical retention volumes of 14 mL, corresponding to an apparent MW of 44 kDa. (C) Comparative thermal melting of H7 and H7-H188A/H197A monitored at 202 nm in CD revealed that the 2nd inflection point was shifted from 57 to 51.5 °C. This destabilizing effect of the His-to-Ala substitutions in the C_L domain allowed us to assign the 1st and 2nd inflection points to the V_L and -C_L domains, respectively. Although the melting curve of H7-H188A/H197A is more cooperative with a single apparent transition, the hypothetical inflection points of the two domains based on the wild-type profile are indicated. (D) H₂O₂ produced by 50 µg/mL H7, H7-H188A/H197A and M7, incubated at different times at 37 °C in 10 mM PBS, pH 7.4. The mean ± SD of fluorescence intensity (FI), n = 6. **** p < 0.0001 vs. H7 WT and mutated H7, one-way Anova and Bonferroni’s post hoc test.

Figure 3

H7 mutant with destabilized C_L domain is less toxic to C. elegans. (A) Dose–response curves reveal the diminished toxicity of H7-H188A/H197A (H7 mut) compared to H7 (WT), as higher concentrations are required to inhibit the pumping rate of worms. Worms were fed for 2 h with different concentrations of WT or mutated H7 suspended in 10 mM PBS, pH 7.4, and the pharyngeal pumping was scored 24 h after the administration. Control worms received vehicle alone (dotted line). Each value is the mean ± SE, n = 30. IC₅₀ was 28.9 and 46.8 µg/mL for WT and mutated H7, respectively, p < 0.01, Student’s t-test. (B) Worms were fed for 2 h with 10 or 100 µg/mL H7 or H7-H188A/H197A, or 100 µg/mL M7 dissolved in 10 mM PBS, pH 7.4, with or without 50 µM Cu (II). Control worms received 10 mM PBS, pH 7.4 with or without 50 µM Cu (II) (vehicle). Pharyngeal pumping was determined 24 h after the administration. Each value is the mean ± SE, n = 20. * p < 0.05, ** p < 0.01 and **** p < 0.001 vs. the corresponding vehicle, °°°° p < 0.001, one-way ANOVA and Bonferroni’s post hoc test.