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

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloid ogenic from nonamyloidogenic light chain proteins has impeded our understan ding of the structural basis of light-chain fibril formation. Moreover, eve n among the subset of human light chains that are amyloidogenic, many prima ry structure differences are found. We compared the thermodynamic stabiliti es of two recombinant kappa 4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V-L from a benign light chain. The a myloidogenic V(L)s were significantly less stable than the benign V-L. Furt hermore, only the amyloidogenic V(L)s formed fibrils under native condition s 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(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabil izing mutations induced fibril formation in vitro. We found that fibril for mation by the benign V-L could be induced by low concentrations of a denatu rant. This indicates that there are no structural or sequence-specific feat ures of the benign V-L that are incompatible with fibril formation, other t han its greater stability. These studies demonstrate that the V-L beta-doma in structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of var iable domain stability is a major driving force in fibril formation.