Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains

Abstract

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant κ4 light-chain variable domains (V_Ls) derived from amyloidogenic light chains with a V_L from a benign light chain. The amyloidogenic V_Ls were significantly less stable than the benign V_L. Furthermore, only the amyloidogenic V_Ls formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V_Ls on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V_L could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V_L that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V_L β-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.