Role of domain interactions in the aggregation of full-length immunoglobulin light chains

Abstract

Amyloid light-chain (LC) amyloidosis is a protein misfolding disease in which the aggregation of an overexpressed antibody LC from a clonal plasma cell leads to organ toxicity and patient death if left untreated. While the overall dimeric architecture of LC molecules is established, with each LC composed of variable (VL) and constant (CL) domains, the relative contributions of LC domain–domain interfaces and intrinsic domain stabilities to protection against LC aggregation are not well understood. To address these topics we have engineered a number of domain-destabilized LC mutants and used solution NMR spectroscopy to characterize their structural properties and intrinsic stabilities. Moreover, we used fluorescence spectroscopy to assay their aggregation propensities. Our results point to the importance of both dimerization strength and intrinsic monomer stability in stabilizing VL domains against aggregation. Notably, in all cases considered VL domains aggregate at least 10-fold faster than full-length LCs, establishing the important protective role of CL domains. A strong protective coupling is found between VL–VL and CL–CL dimer interfaces, with destabilization of one interface adversely affecting the stability of the other. Fibril formation is observed when either the VL or CL domain in the full-length protein is severely destabilized (i.e., where domain unfolding free energies are less than 2 kcal/mol). The important role of CL domains in preventing aggregation highlights the potential of the CL–CL interface as a target for the development of drugs to stabilize the dimeric LC structure and hence prevent LC amyloidosis.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules

“…The isolated V domains from WIL, ALMC2, and 6aJL2 bind to 1 (1 μM) with reduced affinity (Fig. 2D, Left), while JTO-V has K D ligand of 16.5 ± 0.4 μM (mean ± SD; n = 3), similar to that of JTO-FL (20.3 ± 1 μM), consistent with JTO-V being mainly dimeric at this concentration (SI Appendix, Table S2) (22). Eliminating the interchain disulfide bond between the C domains by the C214S mutation reduces the affinity of LCs for 1 (1 μM) ( Fig.…”

“…2C). Recombinant FL λ6-57 LCs form disulfidelinked dimers (4), but their isolated V domains populate an equilibrium between monomers and dimers (4,22,29). The apparent positive cooperativity in the binding of 1 to WIL-V is consistent with induced dimerization of the LC V domain.…”

“…Concentration-dependent NMR chemical shift data indicate that WIL-V has a dimerization equilibrium constant (K D dimer ) of ∼5 mM (SI Appendix, Table S2) (22). Fitting the fluorescence data to a model of binding-induced dimerization constrained by the NMR-derived K D dimer (SI Appendix, SI Materials and Methods) yields an apparent K D ligand of 3.0 ± 1.2 μM for WIL- Fig.…”

“…To further test the hypothesis that 1 binds at the interface between the V domains, we measured binding of 1 to WIL-FL and JTO-FL variants with mutations designed to disrupt the dimer (22). The F98D mutation, which disrupts the V-domain-V-domain interface, rendered binding of 1 (1 μM) undetectable in the presence or absence of the C214 interchain disulfide bond ( Fig.…”

“…We refer to such molecules as kinetic stabilizers, since they reduce the rate at which LCs transiently visit nonnative, aggregation-prone, and protease-sensitive conformations (20). The interfaces between the domains of the LC dimer are an important determinant of stability and aggregation propensity that we and others have identified as potential targets for stabilization (21,22). We therefore seek treatments that prevent newly synthesized and secreted FL LCs from misfolding and aggregating, thus reducing organ proteotoxicity, such that patients can eventually tolerate effective chemotherapy regimens.Using a protease-coupled fluorescence polarization (PCFP) assay that assesses LC kinetic stability, we screened 650,000 small molecules and identified FL LC kinetic stabilizers in four structural classes that protect recombinant and plasma cell-secreted λ6a LCs from endoproteolysis.…”