Elucidating the mechanism of familial amyloidosis- Finnish type: NMR studies of human gelsolin domain 2

Abstract

Familial amyloidosis-Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between beta-strand 4 and alpha-helix 1 and within the loop region following beta-strand 5. Chemical shift differences are noted along the face of alpha-helix 1 that packs onto the beta-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. [(1)H-(15)N] nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

Figure 1

(A) Structure of gelsolin D2 excised from the crystal structure of whole equine gelsolin (11). The secondary elements are a five-stranded β-sheet (strand 1, residues 161–166; strand 2, residues 171–176; strand 3, residues 187–193; strand 4, residues 196–201; strand 5, residues 230–235) and 2 α-helices (helix 1, residues 206–223; helix 2, residues 241–247). The mutation site, Asp-187 (red), is at the N-terminal end of β-strand 3, and the masked cleavage site, Arg-172–Ala-173 (magenta), is in β-strand 2. In full-length gelsolin, β-strand 2 forms additional sheet structure with 2 strands from domain 1 and is not exposed. (B) HSQC chemical shift differences are mapped onto the crystal structure of gelsolin D2. The differences are calculated as: Diff = |(δ_N-WT - δ_N-D187N)|/7 + |(δ_H-WT − δ_H-D187N)|. If the residue is a glycine, the difference between the ¹⁵N chemical shifts is divided by 5. Differences are 0–0.1 (cyan), 0.1–0.2 (yellow), and >0.2 (red). Prolines and exchange-broadened residues are gray. (C) The C_α chemical shift differences are displayed. The differences are 0–0.2 (cyan), 0.2–0.4 (yellow), and >0.4 (red). C_αs that could not be assigned are gray. The results from the relaxation experiments are displayed for WT (D) and D187N (E). Residues that had high or low J_eff(0) values indicative of exchange or increased mobility, respectively, are colored magenta. Also, those residues that are exchanged broadened in the HSQC spectra are colored green (in WT and D187N) or orange (in D187N only). Figs. 1, 3, and 4 were produced by using the program molmol (41).

Figure 2

Results from NMR experiments. The differences in the HSQC (A) and C_α (B) chemical shifts between WT and D187N are displayed. The {¹H-¹⁵N} NOEs are displayed as (I_sat − I_ref)/I_ref for WT (C) and D187N (D). The more negative values are indicative of motion on the nanosecond time scale. The C-terminal tail of D187N begins falling off at Ala-252 compared with Glu-258 in WT, suggesting that six more residues are unstructured in the mutant.

Figure 3

Space-filling model of full-length equine gelsolin. Domain 2 (gray) makes contacts with domains 1, 3, and 6 (black). The cleavage site (magenta) sits in a cleft that is covered by the C-terminal tail of domain 2 (green and orange). In A, the C-terminal tail is present and the portion of the tail with low {¹H-¹⁵N} NOE values in both WT, and D187N is colored green. The additional residues that are believed to be unstructured or in some form of conformational exchange in D187N only are colored orange. In B, the mobile portion of the C-terminal tail in D187N (residues 253–266) has been removed, and the exposed cleavage site is visible. We propose that the C-terminal tail of domain 2 is more unstructured in the D187N mutant, resulting in better access to the masked cleavage site for proteases. The proteolysis fragment can be hydrolyzed further at residue 243, producing the amyloidogenic fragment.

Figure 4

Hydrogen-bonding network connecting the mutation site to residues 235–238. In the equine crystal structure, Asp-187 forms a cross-strand hydrogen bond to Gly-202, most likely stabilizing the mobile Gly. The neighboring Ser-203 has an amide hydrogen that is hydrogen bonded to the side chain 236. This hydrogen bond appears to be the only interaction between the loop region of residues 235–238 with the rest of the protein. The mutation at 187 could destroy this hydrogen-bond network and lead to a conformational transition for residues 235–238 that causes the exchange broadening in the HSQC of D187N.