LC systems studied in this work. The table includes information about the germline, phenotype, method to obtain structure, the SAXS curves, and the radius of gyration derived from the SAXS data for all the model proteins studied in this work.

Comparison of SAXS data and single light chain structures.

Kratky plots of for AL- and MM light chains. The experimental (orange) and theoretical (black) curves (top panels) and the associated residuals (bottom panels) indicate that AL-LC solution behavior deviates from reference structures more than MM-LC. SAXS was measured as follow: H3 measured in bulk (Hamburg), 3.4 mg/ml; H7 measured in bulk (Hamburg), 3.4 mg/ml; H18 measured by SEC-SAXS (ESRF) with the injection concentration of at 2.8 mg/ml; AL55 measured in bulk (ESRF), 2.6 mg/ml; M7 measured in bulk (Hamburg), 3.6 mg/ml; M10 measured by SEC-SAXS with the injection concentration of 6.7 mg/ml (ESRF). Theoretical SAXS curves were calculated using crysol (54). Log-log plots are shown in Fig. S1 in the Supporting Information.

Metainference simulations performed in this work for the 6 systems. For each metainference simulation is reported the simulation time per replica with the number of replicas; the chi-square of the resulting conformational ensemble with the experimental SAXS curve, the range q<0.3 Å is the one used as restraint in the simulation; the average radius of gyration with error estimated by block averaging.

Light chain SAXS driven MD simulations.

(A) Kratky plots and associated residuals (bottom panels) comparing experimental (orange), and theoretical (black) curves obtained by averaging over the metainference ensemble for H3, H7, H18, AL55, M7 and M10, respectively. Theoretical SAXS curves were calculated using crysol (54). (B) Residue-wise root mean square fluctuations (RMSF) obtained by averaging the two Metainference replicates and the two equivalent domains for the six systems studied. The top panel shows data for the variable domains, while the bottom panel shows data for the constant domain. Residues are reported using Chothia and Lesk numbering (51). (C) Schematic representation of two global collective variables used to compare the conformational dynamics of the different systems, namely the distance between the center of mass of the VL and CL dimers and the angle describing the bending of the two domain dimers.

Free Energy Surfaces (FESes) for the six light chain systems under study by Metadynamics Metainference MD simulations.

For each system, the simulations are performed in duplicate. The x-axis represents the elbow angle indicating the relative bending of the constant and variable domains (in radians), while the y-axis represents the distance in nm between the center of mass of the CL and VL dimers. The free energy is shown with color and isolines every 2kBT corresponding to 5.16 kJ/mol. On each FES are represented four regions (green LB-state, red LS-state, blue H-state, and black G-state) highlighting the main conformational states. For each region, a representative structure is reported in a rectangle of the same color.

Populations of the four states shown in Figure 3 resulting from the two independent Metadynamics Metainference simulations performed for each of the 6 LCs. The population of the H state, which we supposed to be a fingerprint specific for AL-LCs, is in bold.

FESes for the four substates identified in Figure 3 in the case of the first H3 Metainference simulation.

The x-axis shows the distance between the centers of mass of the constant domains, while the y-axis shows the distance between the centers of mass of the variable domains. The free energy is shown with color and isolines every 2kBT corresponding to 5.16 kJ/mol.

HDX-MS analysis.

(A) The top panel represents the simplified presentation of the primary structure of an LC including variable domain (VL) and constant domain (CL). The location of β-strands according to Chothia and Lesk (51). (B) The structural mapping and butterfly plot of relative deuterium uptake (Da) of H3. The chain A of H3 structure is colored in a gradient of blue-white-red for an uptake of 0 to 30% at an exchange time of 30 min. The chain B is shown in gray (right hand panel). The butterfly plot showing relative deuterium uptake at all time points from 0.5 to 240 min on a gradient of light to dark blue (left-hand panel). (C, D, E) are the figures corresponding to H7, AL55, and M10, respectively with the same color coding as in figure B. The overall sequence coverage for all proteins was > 90% with a redundancy of > 4.0. The VL-VL domains interface peptides covering amino-acid residues 34-50 are labelled in orange while CL-CL interface region containing 152-180 amino acids are labelled in magenta. Collectively, they form VL-CL interface which is important to define H-state.

Schematic representation summarizing our findings in the context of previous work on the biophysical properties of amyloidogenic light chains.

We propose that the H state is the conformational fingerprint distinguishing AL LCs from other LCs, which together with other features contributes to the amyloidogenicity of AL LCs.