Conformational dynamics in crystals reveal the molecular bases for D76N beta-2 microglobulin aggregation propensity

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.

Conformational stability and dynamics in crystals recapitulate protein behaviour in solution

“…The proteins were extracted and purified following previously published protocols (21). For NMR studies, 13 C, 15 N and 2 H enrichment was obtained as described in (16).…”

“…Simulations data for wt β2m and D76N β2m in crystals were taken from (16), simulations data for wt β2m in solution were taken from (30), where all simulations were run following comparable procedures. D76N β2m simulation in solution were newly performed using the corresponding NMR chemical shifts, following the protocol briefly reported in the following.…”

“…Conformational dynamics of β2m in crystals and in solution were analysed by combining NMR chemical shifts and MD simulations in the theoretical framework of Metainference. In previous works we have characterised the conformational ensembles for wt and D76N β2m in crystal and for wt β2m in solution (16,31). Here, we complement the analysis by collecting NMR chemical shifts in solution for D76N β2m (Fig.…”

“…The amplitude and timescale of conformational exchange processes may be affected by crystal packing, nevertheless they have been shown to directly report on the corresponding motions happening in solution (15). While many of these data have been performed on model crystalline samples, we recently investigated the molecular bases of D76N β2-microglobulin (β2m) pathologic amyloid propensity by studying protein dynamics in crystals (16). Combining solid-state NMR and replica-averaged metadynamics ensemble simulation, we showed that the presence of 'hidden' conformations and increased dynamics in crystals correlates with amyloid formation (16).…”

“…The D76N genetic variant, instead, is responsible for a familial systemic amyloidosis (19), and shows increased aggregation propensity as compared to the wt protein (19,20). The remarkable properties of D76N β2m can be related to the perturbation of a crucial network of weak intramolecular interaction resulting in increased dynamics, lower protein stability and increased amyloidogenicity for a nativelike excited state (16). Conversely, an artificial mutant of the evolutionary conserved Trp60 displays a much lower aggregation propensity (21).…”