A CDR1 aggregation hotspot controls unfolding kinetics and multimolecular aggregation in IGLV2 light chains.

Immunoglobulin light chains (LCs) exhibit diverse aggregation behaviours that depend sensitively on sequence composition and intermolecular interactions. Understanding how specific residues modulate aggregation kinetics remains a key challenge in elucidating the molecular basis of light-chain amyloidosis. Here, we investigate sequence-dependent aggregation using recombinant λ LCs derived from the IGLV2 gene family. Comparison of two closely related LCs differing by only 16 amino acids revealed striking differences in aggregation behaviour under thermal stress. Bioinformatic analysis identified an additional aggregation-prone segment in the CDR1 region of the aggregation-prone M10 variant, associated with residues Ser33 and Tyr34. Rational substitution of these residues (S33D/Y34S) markedly reduced aggregation while leaving the thermal transition temperature largely unchanged (∼53 °C). Differential scanning calorimetry revealed that the wild-type M10 LC unfolds with a significantly lower apparent activation energy (∼290 kJ/mol) compared with the non-aggregating H9 (∼605 kJ/mol) and the stabilised double mutant (∼560 kJ/mol), indicating reduced kinetic stability. Aggregation of unfolded species showed much weaker temperature dependence (Ea ≈ 10-70 kJ/mol) and exhibited strong concentration dependence consistent with a multimolecular association process. Additional experiments suggest that aromatic interactions involving Tyr34 contribute to the stabilisation of intermolecular assemblies. Together, these results establish a quantitative link between local sequence variation in the CDR1 region, kinetic stability of the LC fold, and aggregation propensity, highlighting how targeted mutations can modulate aggregation behaviour in immunoglobulin light chains.