DE-loop mutations affect beta2 microglobulin stability, oligomerization, and the low-pH unfolded form

Abstract

Beta2 microglobulin (beta2m) is the light chain of class-I major histocompatibility complex (MHC-I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of beta2m aggregation, several beta2m mutants have been previously studied. Among these, three single-residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect beta2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild-type (w.t.) beta2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray-ionization mass spectrometry (ESI-MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE-loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, beta2m DE-loop mutations display long-range effects, affecting stability and structural properties of the native protein and its low-pH intermediate. The evidence presented here hints to a crucial role played by the DE-loop in determining the overall properties of native and partially folded beta2m.

Figure 1

Cartoon representation of β2m tertiary structure. The residues mutated in this study (Trp60 and Asp59), the residues forming the disulphide bridge (Cys80 and Cys25), and the residue mainly responsible of intrinsic protein fluorescence (Trp95) are shown in sticks.

Figure 2

Thermal unfolding of w.t. β2m and DE-loop mutants monitored by: (A) near-UV CD at 293 nm; and (B) far-UV CD at 202 nm. In both panels, w.t. β2m unfolding profile is shown in red, W60G in blue, W60V in green, and D59P in black.

Figure 3

Protein oligomerization. Nano-ESI-MS of (A) w.t., (B) W60G, (C) W60V and (D) D59P β2m, at variable protein concentration (in milli-Q water, pH 6.5). The most intense peak of the monomer (•), dimer (○), trimer (Δ) and tetramer (□) is labeled by the corresponding symbol and by the charge state.

Figure 4

Protein unfolding. Nano-ESI-MS of 2.5 μM w.t. β2m, in 10 mM ammonium acetate at variable pH and other denaturing conditions. (A) pH 7.4; (B) pH 2.5; (C) pH 2.5, 50% acetonitrile; (D) pH 2.5, 20 mM DTT (after 24-h incubation at room temperature). Peak envelopes are labeled by the main charge state. The frame in panel D includes peaks that reveal an average-mass shift of 2 Da.

Figure 5

Reduction kinetics. Nano-ESI-MS of equimolar (2.5 μM) mixtures of w.t. (black) and mutant (red) proteins at variable time of incubation in 10 mM ammonium acetate pH 2.5, 20 mM DTT, at room temperature. (A) w.t. and W60G; (B) w.t. and W60V; (C) w.t. and D59P. Frames include peaks that reveal an average-mass shift of 2 Da. Peak envelopes are labeled by the main charge state. Due to the similar molecular weight of w.t. and D59P, the presence of the two components is shown in zoomed images of the 15+ peaks in the insets of panel C.