Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

Abstract

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.

Fig. 1.

The effects of the I56T and D67H mutations on the X-ray structure and NMR chemical shifts of human lysozyme. (A,B) The differences in C_α positions from the X-ray structure of the two variants (Booth et al. 1997). (C,D) The differences in the chemical shifts of the backbone ¹HN resonances at 37°C are shown as black bars (positive values) and gray bars (negative values). (E,F) The differences in the chemical shifts of the backbone ¹⁵N resonances at 37°C are shown. At the top of A and B, the secondary structural elements are shown: helices (bars) and β-sheets (arrows). (A,C,E) I56T minus wild type. (B,D,F) D67H minus wild type.

Fig. 2.

¹H–¹⁵N heteronuclear NOE values for (A) wild-type, (B) I56T, and (C) D67H human lysozymes. NOE values are shown at 20°C (open circles) and 37°C (closed circles). At the top of each figure the secondary structural elements are shown: helices (bars) and β-sheets (arrows). At 20°C, the NOE value of R50 in D67H is 0.79 and is similar to its surrounding residues. At 37°C, however, the measured value is only 0.38. The overlap of resonances in the wild-type protein and I56T variant prevents the measurement of NOEs for this residue in these proteins. We cannot, therefore, account for this apparently low NOE value in D67H at 37°C.

Fig. 3.

Ribbon diagram of the crystal structure of D67H lysozyme produced with Rasmol and the PDB file 1LYY (Booth et al. 1997). The α- and β-domains are in the lower right and upper left sides. The mutation sites, I56T and D67H, are shown in space-filling representations. The molecule is red in regions showing greatly altered chemical shifts (G48–D52 and T60–S82) and blue where reduced NOE values were measured (G72–V74).