Towards an understanding of the structural molecular mechanism of beta(2)-microglobulin amyloid formation in vitro

Abstract

Deriving a complete understanding of protein self-association into amyloid fibrils across multiple distance and time scales is an enormous challenge. At small length scales, a detailed description of the partially folded protein ensemble that participates in self-assembly remains obscure. At larger length scales, amyloid fibrils are often heterogeneous, can form along multiple pathways, and are further complicated by phenomena such as phase-separation. Over the last 5 years, we have used an array of biophysical approaches in order to elucidate the structural and molecular mechanism of amyloid fibril formation, focusing on the all beta-sheet protein, beta(2)-microglubulin (beta(2)m). This protein forms amyloid deposits in the human disease 'dialysis-related amyloidosis' (DRA). We have shown that under acidic conditions beta(2)m rapidly associates in vitro to form amyloid-like fibrils that have different morphological properties, but which contain an underpinning cross-beta structure. In this review, we discuss our current knowledge of the structure of these fibrils, as well as the structural, kinetic and thermodynamic relationship between fibrils with different morphologies. The results provide some of the first insights into the shape of the self-assembly free-energy landscape for this protein and highlight the parallel nature of the assembly process. We include a detailed description of the structure and dynamics of partially folded and acid unfolded species of beta(2)m using NMR, and highlight regions thought to be important in early self-association events. Finally, we discuss briefly how knowledge of assembly mechanisms in vitro can be used to inform the design of therapeutic strategies for this, and other amyloid disorders, and we speculate on how the increasing power of biophysical approaches may lead to a fuller description of protein self-assembly into amyloid in the future.

Fig. 1. Ribbon diagram of the crystal structure of the MHC-I complex

(a); the structure of β₂m in the MHC complex (b); the crystal structure of monomeric human β₂m (c); the solution structure of monomeric human β₂m (d). The structures were taken from 1DUZ (for (a) and (b) [75]), 1LDS (for (c) [17]) and 1JNJ (for (d) [16]). Individual subunits in the MHC heavy chain (α1 – 3), as well as individual β-strands in native β₂m, (A–G) are shown. The figures were drawn using the program MOLMOL [76].

Fig. 2

Schematic diagram outlining events involved in the development of DRA. The molecular mechanism of self-association in vivo is unknown. Several biological factors may be involved in the development of the disease. Lower right image: an anterior view of a DRA patient imaged using ¹¹¹In-β₂m scintigraphy showing the accumulation of β₂m amyloid deposits in the elbows and wrists (reproduced with permission from reference [77]).

Fig. 3. AFM images of the different fibril morphologies formed from β₂m at pH 3.5 under different solution conditions

(a) long-straight (LS); (b) worm-like (WL) and (c) rod-like (RL). RL fibrils were formed in buffer containing 25 mM sodium phosphate and 25 mM sodium acetate, pH 3.5 (no agitation), whilst WL fibrils were formed under identical conditions but containing 200 mM NaCl (no agitation), and LS fibrils were formed in 5 mM ammonium acetate and 5 mM ammonium formate buffer at pH 3.5 with agitation (200 rpm). All images were taken after an incubation time of at least 2 weeks at 37 °C.

Fig. 4

At low pH, β₂m assembly can be depicted on an energy diagram involving at least two competing pathways [30], the shape of which can be manipulated by solution conditions (different coloured lines). At the molecular level, how these pathways are ‘gated’ is currently unknown.

Fig. 5

Populations of native, partially folded and acid unfolded molecules of β₂m as determined by fitting the charge state distribution in ESI mass spectra to a series of Gaussian distributions at several pH values [58]. Data are shown for wild-type β₂m (black) and two variants, one with an amino acid substitution in native strand-A (V9A, grey) and one with an amino acid substitution in the core of the protein (F30A, red).

Fig. 6

¹H– ¹⁵N HSQC spectra of β₂m obtained in 90% (v/v) H₂O, 10% (v/v) D₂O at pH 7.0 (a); pH 3.6 (b) and pH 2.5 (c) at 37 °C recorded at 500 MHz.

Fig. 7

Titration of partially unfolded β₂m (pH 3.6) and acid unfolded β₂m (pH 2.5) with urea at 25 °C. In each case, the intensity of individual residues is shown as a function of the concentration of urea (0 – 3 M).

Fig. 8. Residue specific NMR transverse relaxation rates for wild-type β₂m in water at pH 2.5, 25 °C, obtained at 500 MHz

(a). The black line is a fit to a model of a random coil containing a single disulphide bond, and the grey line is a fit to the observed rates, showing significant residual structure in the core of the protein, specifically in the residues that comprise the native E-strand (residues 60–71). The position of β-strands in native β₂m is shown above the plot and residues that are aromatic are marked as a grey dot. Thioflavin-T fluorescence of fibrils formed from β₂m and different peptides corresponding to individual strands of β₂m incubated at pH 3.6 in 400 mM NaCl [63] (b). Prediction of the aggregation propensity of the amino acid sequence of β₂m using the TANGO algorithm [64] (c). The structure of residues 60 – 70 in native β₂m (equivalent to the native E strand) showing the high content of aromatic residues in this region is shown in (d). The image was drawn using MOLMOL [76].

Fig. 9

Schematic diagram of possible models for β₂m amyloid fibril formation in vitro. As indicated, β₂m dissociates from the MHC heavy chain and under amyloidogenic conditions, is destabilised. Possible conformational changes which increase the propensity to self-assemble are depicted in the figure as (a) population of one or more partially folded conformations; (b) the loss of the β-bulge in strand D; (c) the displacement of the edge-strands; (d) formation of extensively unfolded structures.