Stabilizing an amyloidogenic λ6 light chain variable domain

Luna-Martínez, Oscar D.; Hernández-Santoyo, Alejandra; Villalba-Velázquez, Myriam I.; Sánchez-Alcalá, Rosalba; Fernández‐Velasco, D. Alejandro; Becerril, Baltazar

doi:10.1111/febs.14265

Cited by 10 publications

(19 citation statements)

References 65 publications

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“…The model protein 6aJL2, a recombinant (r) V L protein within the λ6 subgroup, has a particularly amyloidogenic mutant 6aJL2‐R24G (one should note that here we use continuous numbering of the amino‐acid residues along the protein; an alternative numbering scheme in which Gly24 becomes Gly25 exists). Both proteins have been comprehensively characterized by in vitro fibrillization, and their monomer structures have been determined by X‐ray crystallography and solution‐state NMR .…”

Section: Figurementioning

confidence: 99%

A Substantial Structural Conversion of the Native Monomer Leads to in‐Register Parallel Amyloid Fibril Formation in Light‐Chain Amyloidosis

et al. 2019

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Amyloid light‐chain (AL) amyloidosis is a rare disease in which plasma‐cell‐produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid‐associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid‐state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show β‐strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially 15N:13C‐labeled samples, that the β‐strands are stacked in‐register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.

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Section: Figurementioning

confidence: 99%

A Substantial Structural Conversion of the Native Monomer Leads to in‐Register Parallel Amyloid Fibril Formation in Light‐Chain Amyloidosis

et al. 2019

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“…In this work, we determined a crystal structure by molecular replacement of recombinant 6aJL2-R24G at 2.0 Å resolution crystallized in a new condition, with two subunits in the asymmetric unit. The structure analysis shows that this structure, although crystallized in a different space group, by crystal symmetry generates octamers in a helicoidal arrangement, which is also found in structures of protein variants with PDB entries 5C9K, 2W0K, 5IR3 and 1PW3 among others [16,18,19]. This octameric assembly has been proposed as an important early stage in amyloid fibril aggregation [16].…”

Section: Introductionmentioning

confidence: 67%

“…The structure analysis shows that this structure, although crystallized in a different space group, by crystal symmetry generates octamers in a helicoidal arrangement, which is also found in structures of protein variants with PDB entries 5C9K, 2W0K, 5IR3 and 1PW3 among others [16,18,19]. This octameric assembly has been proposed as an important early stage in amyloid fibril aggregation [16]. However, other crystal structures of protein variants do not make that octameric assembly in a helicoidal arrangement (PDB entries: 3B5G, 3BDX [18], 2W0L [Unpublished], 1CD0 and 2CD0 [20] among others).…”

Section: Introductionmentioning

confidence: 70%

“…of 2.0 Å using 106 Cα atoms and 0.52 Å using 111 Cα atoms, respectively. Also, 6aJL2-R24G was superposed with the 6aJL2-R24G NMR structure (PDB ID 2MKW, model 1) and the 6aJL2-R24G crystal structure (PDB ID 5C9K, [16]). The r.m.s.d of the structural alignments are 1.16 Å using 109 Cα residues and 0.19 Å using 111 Cα residues for entries 2 MKW and 5C9K, respectively, showing that the global structures are almost identical, particularly in the crystal structures.…”

Section: Resultsmentioning

confidence: 99%

“…In vitro studies suggest that this mutation eliminates important interactions, including a cation-pi with Phe2, an ionic interaction with Asp93 and some H-bonds [15]. 6aJL2-R24G first crystal structure shows that in the absence of Arg24, Phe2 is reoriented to the inside of the upper hydrophobic core and a cavity is formed around the complementary determining region 1 (CDR1) [16]. Molecular dynamic simulations find differences between 6aJL2 and 6aJL2-R24G in their dynamical signatures that explain the variant increased tendency to form amyloids [17].…”

Section: Introductionmentioning

confidence: 99%

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

Rudino‐Pinera

Pelaez-Aguilar

Amero

et al. 2019

Biochemistry and Biophysics Reports

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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.

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Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains

Rottenaicher

Weber

Rührnößl

et al. 2021

Journal of Biological Chemistry

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Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody V L domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient V L domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient V L domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the V L domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.

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Stabilizing an amyloidogenic λ6 light chain variable domain

Abstract: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 5IR3 and 5C9K.

Cited by 10 publications

References 65 publications

A Substantial Structural Conversion of the Native Monomer Leads to in‐Register Parallel Amyloid Fibril Formation in Light‐Chain Amyloidosis

A Substantial Structural Conversion of the Native Monomer Leads to in‐Register Parallel Amyloid Fibril Formation in Light‐Chain Amyloidosis

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

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains

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