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Published on: September 17, 2017
Decoding the Structural Bases of D76N ß2-Microglobulin High Amyloidogenicity through Crystallography and Asn-Scan
Matteo de Rosa1, Alberto Barbiroli2, Sofia Giorgetti3
1Dipartimento di Bioscienze, Università di Milano, Via Celoria 26, 20133, Milano, Italy.
Abstract:
D76N is the first natural variant of human β-2 microglobulin (β2m) so far identified. Contrary to the wt protein, this mutant readily forms amyloid fibres in physiological conditions, leading to a systemic and severe amyloidosis. Although the Asp76Asn mutant has been extensively characterized, the molecular bases of its instability and aggregation propensity remain elusive. In this work all Asp residues of human β2m were individually substituted to Asn; D-to-N mutants (D34N, D38N, D53N, D59N, D96N and D98N) were characterised in terms of thermodynamic stability and aggregation propensity. Moreover, crystal structures of the D38N, D53N, D59N and D98N variants were solved at high-resolution (1.24-1.70 Å). Despite showing some significant variations in their thermal stabilities, none showed the dramatic drop in melting temperature (relative to the wt protein) as observed for the pathogenic mutant. Consistently, none of the variants here described displayed any increase in aggregation propensity under the experimental conditions tested. The crystal structures confirmed that D-to-N mutations are generally well tolerated, and lead only to minor reorganization of the side chains in close proximity of the mutated residue. D38N is the only exception, where backbone readjustments and a redistribution of the surface electrostatic charges are observed. Overall, our results suggest that neither removing negative charges at sites 34, 38, 53, 59, 96 and 98, nor the difference in β2m pI, are the cause of the aggressive phenotype observed in D76N. We propose that the dramatic effects of the D76N natural mutation must be linked to effects related to the crucial location of this residue within the β2m fold.
Insights
The D76N mutation in human beta-2 microglobulin (β2m) causes severe amyloidosis. Substituting other Asp residues to Asn did not replicate this severe amyloidosis, suggesting D76N
Area of Science:
- Biochemistry
- Structural Biology
- Protein Misfolding Diseases
Background:
- Human beta-2 microglobulin (β2m) is crucial for immune function.
- The natural D76N variant of β2m readily forms amyloid fibers, causing severe systemic amyloidosis.
- The molecular basis for the instability and aggregation of the D76N mutant remains unclear.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the instability and aggregation of the D76N β2m variant.
- To determine if substituting other aspartic acid (Asp) residues to asparagine (Asn) in β2m mimics the pathogenic phenotype of D76N.
- To elucidate the structural and thermodynamic consequences of D-to-N mutations in β2m.
Main Methods:
- Site-directed mutagenesis to create D-to-N mutants (D34N, D38N, D53N, D59N, D96N, D98N).
- Characterization of thermodynamic stability using melting temperature assays.
- Assessment of aggregation propensity under physiological conditions.
- High-resolution crystal structure determination of selected D-to-N variants.
Main Results:
- None of the generated D-to-N mutants exhibited a significant decrease in thermal stability or increased aggregation propensity compared to wild-type β2m.
- Crystal structures revealed that D-to-N mutations are generally well-tolerated, causing minor side-chain reorganizations.
- The D38N mutant showed notable backbone adjustments and altered surface electrostatic charge distribution.
Conclusions:
- The aggressive amyloidosis phenotype of D76N is unlikely due to the removal of negative charges at the mutated sites or changes in the isoelectric point (pI) of β2m.
- The specific location of Asp76 within the β2m fold is critical for the pathogenic effects of the D76N mutation.
- Further structural and functional studies are needed to fully understand the molecular basis of D76N-induced amyloidosis.
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