Abstract
Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process. A prototypic example is the D76N mutant of beta-2 microglobulin (β2m) that displays an aggressive aggregation propensity. Here we investigate the dynamics of β2m by X-ray crystallography, solid-state NMR, and molecular dynamics simulations to unveil the effects of the D76N mutation. Taken together, our data highlight the presence of minor disordered substates in crystalline β2m. The destabilization of the outer strands of D76N β2m accounts for the increased aggregation propensity. Furthermore, the computational modeling reveals a network of interactions with residue D76 as a keystone: this model allows predicting the stability of several point mutants. Overall, our study shows how the study of intrinsic dynamics in crystallo can provide crucial answers on protein stability and aggregation propensity. The comprehensive approach here presented may well be suited for the study of other folded amyloidogenic proteins.
Highlights
Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process
D76N β2m rapidly converts into non-soluble oligomers and amyloid fibrils in solution, the intrinsic dynamics of both wt and mutant forms of the protein can be studied in the crystals using solid-state Nuclear magnetic resonance (NMR)
In 2012 the β2m D76N mutant was reported to be responsible for an aggressive form of systemic amyloidosis with large fibrillar deposits in internal organs
Summary
Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process. Common effects of point mutations are structural perturbations of the backbone, the impairment of the hydrophobic core or the introduction of hydrophobic patches on the protein surface, with potentially very drastic outcomes involving a marked change in size or in the chemical properties[1]. None of these effects can explain the enhanced reactivity of D76N β2m. D76N β2m rapidly converts into non-soluble oligomers and amyloid fibrils in solution, the intrinsic dynamics of both wt and mutant forms of the protein can be studied in the crystals using solid-state NMR (ssNMR). The wealth of crystallographic data available on β2m and the inherent protection against aggregation makes MAS NMR a technique of choice for a comparative study of the dynamics of wt and D76N β2m
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