A residue-specific shift in stability and amyloidogenicity of antibody variable domains.

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (VL) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of VL domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the VL domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol(-1), which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in VL domains of the κ family and not in VLλ or in VH domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.

A Stable Mutant Predisposes Antibody Domains to Amyloid Formation through Specific Non-Native Interactions.

The aggregation of mostly antibody light chain variable (VL) domains into amyloid fibrils in various tissues is the main cause of death in systemic amyloid light chain amyloidosis. Point mutations within the domain are important to shift the VL into the fibrillar pathway, but why and how only some site-specific mutations achieve this still remains elusive. We show here that both destabilizing and surprisingly stable mutants readily predispose an amyloid-resistant VL domain to amyloid formation. The decreased thermodynamic stability of the destabilizing mutant results in the accumulation of non-native intermediates that readily populate the amyloid state. Interestingly, the stable mutants establish site-specific non-native interactions with especially nearby serine/threonine residues that unexpectedly do not affect the folding behavior of the VL domain but rather readily induce and stabilize the fibril structure, a previously unrecognized mechanism. These findings provide a new concept for the molecular mechanism of amyloid fibril formation.

The Antibody Light-Chain Linker Is Important for Domain Stability and Amyloid Formation.

The association of light chains (LCs) and heavy chains is the basis for functional antibodies that are essential for adaptive immune responses. However, in some cases, LCs and especially fragments consisting of the LC variable (VL) domain are pathologically deposited in fatal aggregation diseases. The two domains of the LC are connected by a highly conserved linker. We show here that, unexpectedly, the linker residue Arg108 affects the conformational stability and folding of both VLκ and LC constant (CLκ) domains. Interestingly, the extension of VL by Arg108 results in its resistance to amyloid formation, which suggests that the nature of the truncation of the LC plays a crucial role in disease progression. Increased solvation due to the exposed charged C-terminal Arg108 residue explains its stabilizing effects on the VL domain. For the CL domain, the interaction of N-terminal loop residues with Arg108 is important for the integrity of the domain, as the disruption of this interaction results in fluctuation, partial opening of the protein's interior and the exposure of hydrophobic residues that destabilize the domain. This establishes new principles for antibody domain architecture and amyloidogenicity.