Crystal structure of 6aJL2-R24G light chain variable domain: Does crystal packing explain amyloid fibril formation?

Abstract

Light chain amyloidosis is one of the most common systemic amyloidosis, characterized by the deposition of immunoglobulin light variable domain as insoluble amyloid fibrils in vital organs, leading to the death of patients. Germline λ6a is closely related with this disease and has been reported that 25% of proteins encoded by this germline have a change at position 24 where an Arg is replaced by a Gly (R24G). This germline variant reduces protein stability and increases the propensity to form amyloid fibrils. In this work, the crystal structure of 6aJL2-R24G has been determined to 2.0 Å resolution by molecular replacement. Crystal belongs to space group I2 1 2 1 2 1 (PDB ID 5JPJ) and there are two molecules in the asymmetric unit. This 6aJL2-R24G structure as several related in PDB (PDB entries: 5C9K, 2W0K, 5IR3 and 1PW3) presents by crystal packing the formation of an octameric assembly in a helicoidal arrangement, which has been proposed as an important early stage in amyloid fibril aggregation. However, other structures of other protein variants in PDB (PDB entries: 3B5G, 3BDX, 2W0L, 1CD0 and 2CD0) do not make the octameric assembly, regardless their capacity to form fibers in vitro or in vivo . The analysis presented here shows that the ability to form the octameric assembly in a helicoidal arrangement in crystallized light chain immunoglobulin proteins is not required for amyloid fibril formation in vitro . In addition, the fundamental role of partially folded states in the amyloid fibril formation in vitro , is not described in any crystallographic structure published or analyzed here, being those structures, in any case examples of proteins in their native states. Those partially folded states have been recently described by cryo-EM studies, showing the necessity of structural changes in the variants before the amyloid fiber formation process starts.

Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G

“…This mutation is present in 25% of all amyloid-associated λ6 LC cases . 6aJL2-R24G protein is less stable and more prone to form amyloid fibers in vitro than the germline protein 6aJL2, while maintaining a similar three-dimensional structure. , 6aJL2-R24G is a β-sandwich domain formed by 111 residues and has a conserved tryptophan residue at position 36, which is quenched in the native state by the nearby conserved disulfide bridge …”

Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains

“…The native structure of 6aJL2 and 6aJL2-R24G has been determined by X-ray crystallography − and solution nuclear magnetic resonance (NMR); both structures were found to be very similar, suggesting that the different amyloid propensities are not due to a structural element. 6aJL2 has 111 residues that adopt a β-sandwich formed by two β-sheets held together by a disulfide bond.…”

“…One sheet includes strands A, B, D, and E, while the other contains strands C, C′, F, and G. In addition, it has three complementarity-determining regions (CDRs) (Figure ). , …”

“…One sheet includes strands A, B, D, and E, while the other contains strands C, C′, F, and G. In addition, it has three complementarity-determining regions (CDRs) (Figure 1). 38,39 The equilibrium and kinetic properties of unfolding as well as the in vitro fibril formation of these two proteins have been studied under different experimental conditions using chaotropic agents (urea and GdnCl), metal ions, and changes in temperature or pH. 12,32,33,35,40,41 In addition, the dynamics of 6aJL2 has been studied by NMR and computer simulations, 37,42 where it was found that strands B, C, and E−G were the most stable.…”

Antibodies gone bad – the molecular mechanism of light chain amyloidosis

“…Additional factors that play a key role in AL amyloidosis are the population of partially folded intermediate states and transient, non-native interactions which allow assembly into amyloid fibrils [105,[137][138][139]. In comparison to their non-amyloidogenic counterparts, these variants more readily transition into a conformation that differs from the native state [71,140,141].…”