A Simulated Intermediate State for Folding and Aggregation Provides Insights into ΔN6 β2-Microglobulin Amyloidogenic Behavior (original) (raw)
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Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin
Journal of Biological Chemistry, 2013
Background: We recently discovered the first natural human  2-microglobulin variant, D76N, as an amyloidogenic protein. Results: Fluid flow on hydrophobic surfaces triggers its amyloid fibrillogenesis. The ␣-crystallin chaperone inhibits variantmediated co-aggregation of wild type  2-microglobulin. Conclusion: These mechanisms likely reflect in vivo amyloidogenesis by globular proteins in general. Significance: Our results elucidate the molecular pathophysiology of amyloid deposition. Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human  2-microglobulin ( 2 m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N  2 m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type  2 m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillo-genesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.
The role of conformational flexibility in β 2 ‐microglobulin amyloid fibril formation at neutral pH
Rapid Communications in Mass Spectrometry, 2012
RATIONALE: Amyloid formation is implicated in a number of human diseases. b 2-microglobulin (b 2 m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and b 2 m amyloid formation at physiological pH has been investigated. METHODS: HDX-ESI-MS was used to study the conformational dynamics of b 2 m. Protein engineering, or the addition of Cu 2+ ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of b 2 m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further. RESULTS: ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type b 2 m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation. CONCLUSIONS: The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of b 2 m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from b 2 m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.
Biochimica Et Biophysica Acta-proteins and Proteomics, 2005
Deriving a complete understanding of protein self-association into amyloid fibrils across multiple distance and time scales is an enormous challenge. At small length scales, a detailed description of the partially folded protein ensemble that participates in self-assembly remains obscure. At larger length scales, amyloid fibrils are often heterogeneous, can form along multiple pathways, and are further complicated by phenomena such as phase-separation. Over the last 5 years, we have used an array of biophysical approaches in order to elucidate the structural and molecular mechanism of amyloid fibril formation, focusing on the all h-sheet protein, h 2 -microglubulin (h 2 m). This protein forms amyloid deposits in the human disease Fdialysis-related amyloidosis_ (DRA). We have shown that under acidic conditions h 2 m rapidly associates in vitro to form amyloid-like fibrils that have different morphological properties, but which contain an underpinning cross-h structure. In this review, we discuss our current knowledge of the structure of these fibrils, as well as the structural, kinetic and thermodynamic relationship between fibrils with different morphologies. The results provide some of the first insights into the shape of the self-assembly free-energy landscape for this protein and highlight the parallel nature of the assembly process. We include a detailed description of the structure and dynamics of partially folded and acid unfolded species of h 2 m using NMR, and highlight regions thought to be important in early self-association events. Finally, we discuss briefly how knowledge of assembly mechanisms in vitro can be used to inform the design of therapeutic strategies for this, and other amyloid disorders, and we speculate on how the increasing power of biophysical approaches may lead to a fuller description of protein self-assembly into amyloid in the future. D
Journal of Molecular Biology, 2008
Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human β 2 -microglobulin (β 2 m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type β 2 m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the β 2 m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29-51 and 58-79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for β 2 m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62-70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.
Journal of Molecular Biology, 2003
Current data suggest that globular domains may form amyloids via different mechanisms. Nevertheless, there are indications that the initiation of the process takes place invariably in the less stable segments of a protein domain. We have studied the sequence and structural conservation of b 2-microglobulin that deposits into fibrils in dialysisrelated amyloidosis. The dataset includes 51 high-resolution nonredundant structures of the antibody constant domain-like proteins (C1) and 132 related sequences. We describe a set of 30 conserved residues. Among them, 23 are conserved structurally, 16 are conserved sequentially and nine are conserved both sequentially and structurally. Strands A (12-18), G (91-95) and D (45-55) are the less conserved and stable segments of the domain, while strands B (22-28), C (36-41), E (62-70) and F (78-83) are the conserved and stable segments. We find that the conserved residues form a cluster with a network of interactions. The observed pattern of conservation is consistent with experimental data including H/D exchange, urea denaturation and limited proteolysis that suggest that strands A and G do not participate in the amyloid fibril. Additionally, the low conservation of strand D is consistent with the observation that this strand may acquire different conformations as seen in crystal structures of bound and isolated b 2-microglobulin. We used a docking technique to suggest a model for a fibril via stacking of b 2-microglobulin monomers. Our analysis suggests that the favored monomer building block for fibril elongation is the conformation of the isolated b 2-microglobulin, without the b-bulge on strand D and without strands A and G participating in the fibril b-sheet structure. This monomer retains all the conserved residues and their network of interactions, increasing the likelihood of its existence in solution. The inter-strand interaction between the two (monomer) building blocks forms a new continuous b-sheet such that addition of monomers results in a fibril model that has the characteristic cross-b structure.
Biology, 2021
β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, th...
Partially Unfolded States of β 2 -Microglobulin and Amyloid Formation in Vitro †
Biochemistry, 2000
Dialysis-related amyloidosis (DRA) involves the aggregation of 2 -microglobulin ( 2 m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric 2 m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are ∼10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric 2 m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK a of 4.7 destabilizes native 2 m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional 1 H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for 2 m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies. † We acknowledge with thanks financial support from The University of Leeds, The Wellcome Trust, and the BBSRC. V.J.M. and N.M.K. are supported by the BBSRC, A.P.K.