Biochemical and biophysical comparison of human and mouse beta‐2 microglobulin reveals the molecular determinants of low amyloid propensity (original) (raw)

The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta-2 microglobulin (β2m), which, when accumulated in patients, gives rise to dialysis-related amyloidosis. Interestingly, although the physiologic concentration of β2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine β2m not only displays a lower amyloid propensity both in vivo and in vitro, but also inhibits the aggregation of human β2m in vitro. Here we compared human and murine β2m for their aggregation propensity, ability to form soluble oligomers, stability, three-dimensional structure and dynamics. Our results indicate that murine β2m low-aggregation propensity is due to two concomitant aspects: the low aggregation propensity of its primary sequence combined with the absence of high-energy amyloidcompetent conformations under native conditions. The identification of the specific properties Accepted Article This article is protected by copyright. All rights reserved. determining the low aggregation propensity of mouse β2m will help delineate the molecular risk factors which cause a folded protein to aggregate. This article is protected by copyright. All rights reserved. energy obtained for hβ2m resembles that previously determined by replica-averaged metadynamics simulations on a slightly different sequence [18, 63]. The main differences being the lack of a more 'crystal'-like high-energy state and the presence of a slightly more disordered, high-energy, state. This latter was not sampled in a former work [18] because the sampling was not allowed in that region, but was more recently observed by solid state NMR and replica-averaged metadynamics simulations [20]. Circular dichroism Thermal stability experiments were performed in triplicate using three independent batches of protein and were monitored in the far-UV region using a J-810 spectropolarimeter (JASCO Corp., Tokyo, Japan) equipped with a Peltier system for temperature control. The protein concentration was 0.1 mg/mL in 50 mM sodium phosphate pH 7.4. The temperature ramps were carried out from 20 to 95 °C (temperature slope 50 °C/hour) in a 0.1 cm path length cuvette and monitored at 202 nm wavelength. Tm was calculated as the first-derivative minimum of the traces. Spectra before and after unfolding ramp were recorded (260-190 nm). All three β2m variants considered in this work display an irreversible unfolding under the tested conditions. Accession number Atomic coordinates and structure factors for mβ2m have been deposited at the Protein Data Bank, with accession code 6I8C.