LC systems studied in this work. For each LC in the table are reported the germline, the phenotype, the structure or the method used to obtain one, the agreement between the structure and the SAXS curves, and the radius of gyration derived from the SAXS data.

SAXS measurements for AL- and M light chains. Kratky plots comparing experimental (orange), and theoretical (black) curves and associated residuals (bottom panels) indicate that H LC solution behavior deviates from reference structures more than M LC. (A) H3 measured in bulk (Hamburg), 3.4 mg/ml. (B) H7 measured in bulk (Hamburg), 3.4 mg/ml. (C) H18 measured by online SEC-SAXS (ESRF), starting at 2.8 mg/ml. (D) AL55 measured in bulk (ESRF), 2.6 mg/ml. (E) M7 measured in bulk (Hamburg), 3.6 mg/ml. (F) M10 measured by online SEC-SAXS starting at 6.7 mg/ml (ESRF).

(A) 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 numbering (49). (B) 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.

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.

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, red, blue, and black rectangles) highlighting their main features. For each region, a representative structure is reported.

Free energy surfaces 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. 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 (49). The middle panel represents the relative HDX butterfly plots of H3, H7, AL55, and M10 proteins. The peptides showing significantly higher deuterium uptake are labeled on their respective peaks. The peptide from residues 34-50 in AL-LCs and 54-70 in M-LC are labeled in orange. The lower panel represents the structural mapping of the selected peptides showing the highest deuterium uptakes using PyMOL. The VL-VL and VL-CL interfaces covering residues 34-50 and residues 161-180 are pointed with dark orange and light orange arrows, respectively.

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.