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Article Dans Une Revue Proceedings of the National Academy of Sciences of the United States of America Année : 2022

The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis

Résumé

Abstract: Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using β2-microglobulin (β2m) as a model system, focusing on D76N-β2m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting β2m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite β-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-β2m) we show that residue 76 has a unique ability to drive β2m aggregation in vivo and in vitro. Using a randomly mutated D76N-β2m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-β2m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state. Significance: Protein aggregation is a major problem for human health. However, our understanding of how folded proteins aggregate into amyloid lags behind. Using the tripartite β-lactamase assay (TPBLA) with our test protein, β 2 -microglobulin (β 2 m), we show the ability to differentiate the behavior of single-point variants and highlight the remarkable sensitivity to the identity of the residue at position 76. After evolving the aggregation-prone protein, D76N-β 2 m, the only mutations able to improve D76N-β 2 m behavior in vivo involve residues in a single 7-residue sequence of the protein. Further characterization in vitro shows that a single-point mutant in this region can abolish D76N-β 2 m aggregation.

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Dates et versions

hal-03779985 , version 1 (18-09-2022)

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N. Guthertz, R. van der Kant, R. Martinez, Y. Xu, C. Trinh, et al.. The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119 (22), pp.e2200468119. ⟨10.1073/pnas.2200468119⟩. ⟨hal-03779985⟩
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