Domain Interactions Determine the Amyloidogenicity of Antibody Light Chain Mutants.

In antibody light chain amyloidosis (AL), mutant light chains (LCs) or their variable domains (VLs) form fibrils, which accumulate in organs and lead to their failure. The molecular mechanism of this disease is still poorly understood. One of the key open issues is whether the mutant VLs and LCs differ in fibril formation. We addressed this question studying the effects of the VL mutations S20N and R61A within the isolated VL domain and in the full-length LC scaffold. Both VL variants readily form fibrils. Here, we find that in the LC context, the S20N variant is protected from fibril formation while for LC R61A fibril formation is even accelerated compared to VL R61A. Our analyses revealed that the partially unfolded state of the VL R61A domain destabilizes the CL domain by non-native interactions, in turn leading to a further unfolding of the VL domain. In contrast, the folded mutant VL S20N and VL wt form native interactions with CL. These are beneficial for LC stability and promote amyloid resistance. Thus the effects of specific mutations on the VL fold can have opposing effects on LC domain interactions, stability and amyloidogenicity.

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16INK4A.

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-ß sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.

The Antibody Light-Chain Linker Regulates Domain Orientation and Amyloidogenicity.

The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (VL) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the VL and CL domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of VL and CL to each other are of major importance for maintaining the structural integrity of the full-length LC.

Physical basis of amyloid fibril polymorphism.

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the ß-sheet twist, as well as peptide-peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-ß fibrils, and suggest that a small number of physical parameters control the observed fibril architectures.

MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors.

Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the ß-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS), using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains.

Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs.

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.

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.