STRUCTURAL CHARACTERIZATION OF THE EARLY STAGES OF THE BETA 2 MICROGLOBULIN AMYLOIDOSIS

ABSTRACT. Background. β-2 microglobulin (β2m) is an amyloidogenic protein responsible for dialysis related amyloidosis in man, which result in the deposition of β2m amyloid fibrils at a skeletal level. β2m is a 99 residue protein formed by two β-sheets linked by a disulphide bond. In the early stages of fibril formation, β2m associate into dimers and higher order oligomers that are structurally poorly characterized due to their transient nature. Furthermore, the aggregation properties of β2m are affected by its fold-stability. In particular, the DE loop region, which connect the D- and the E- strands, has been reported to be crucial for the β2m fold-stability. Results. Four monomeric β2m cysteine mutants (S20C, E50C, W60C and S88C) were produced and their correspondent disulphide-linked homodimers were prepared (DIMC20, DIMC50, DIMC60 and DIMC88). The aggregation properties, the crystallogenesis and the oligomerisation state in solution were tested for each β2m homodimer. DIMC20, DIMC50 and DIMC88 form amyloid fibrils, crystals and display a varying mixtures of dimeric and tetrameric species in solution, while DIMC60 is not amyloidogenic and is purely dimeric in solution. DIMC20 and DIMC50 X-ray structures (2.45 Å and 2.7 Å resolution, respectively) shared a non-covalent D-D strand interface that mediate the formation of a tetrameric assembly in both DIMC20 and DIMC50. Moreover, DIMC20 and DIMC50 in solution can catalyse the w.t. β2m fibrils formation in the absence of fibril seeds at pH 7.4, strongly suggesting that the D-D strand interface is involved in the early stages of β2m amyloid aggregation. In order to further characterize the role of the DE loop in β2m fold-stability, a K58P-W60G β2m mutant was produced and purified. The K58P-W60G β2m mutant showed improved thermal and chemical stability and a faster folding compared to the w.t. β2m. The crystal structure of the K58P-W60G β2m mutant (1.25 Å resolution) showed that the internal disulphide bond was severed as reported by Electrospray ionization-mass spectrometry spectra, which display that a fraction of the K58P-W60G β2m mutant has a reduced disulphide bond. These data suggest a stabilizing role of Pro58 and stress the importance of the DE loop on the biophysical properties of the β2m.