The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity

Abstract

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.

Keywords: X-ray crystallography; ion mobility mass spectrometry; nuclear magnetic resonance spectroscopy.

Fig. 1.

Crystal structures of cABC and cHSP27. (A) cABC crystallizes as a dimer of dimers, with one C-terminal peptide of sequence ERTIPITRE (red) bound to each monomer. (B) Two Ig-like cABC monomers assemble into a dimer through pairwise and antiparallel interactions between extended β6+7 strands. In this structure the dimer is found in the AP_III register. (Inset) The palindromic C-terminal peptide binds to a hydrophobic groove between the β4 and β8 strands, in an antiparallel direction to the β8 strand. The N-to-C direction is illustrated by the yellow arrow. (C) The crystal structure of cHSP27 reveals a dimer similar to cABC, rich in β-sheet structure and with C-terminal peptides bound. (Inset) cHSP27, however, is in the AP_II register, with C137 (thiol colored in yellow) located about a twofold axis at the dimer interface. C137 is reduced in the structure because of the presence of reductant during crystallization but can be oxidized readily (Fig. 4).

Fig. 2.

Structural differences between cHSP27 and cABC. (A) Sequence alignment of the two domains colored to highlight differences in amino acid composition (strongly dissimilar, red; dissimilar, orange; weakly dissimilar, yellow; Materials and Methods). The two domains are clearly highly similar (54.7% identity), although differences are distributed throughout the sequence. (B) Mapping the disparate residues on the cABC (Left) and cHSP27 (Right) structures (colored as in A) shows that they are spread over the entire structure. (C) Expansions of the boxed regions in B reveal a charge network formed by residues E99, H101, E117, and H119 in cABC (Left) that is absent in cHSP27 (Right) where the equivalent residues are E119, T139, T121, and C137.

Fig. 3.

NMR and IM reveal AP_II as the dominant registration state for cABC in solution. (A) Crystal structure of cABC_E117C in which the introduced cysteine acts to lock the domain into a dimer in the AP_II register. The Inset shows the disulfide bond formed between two monomers. The overall structure of this engineered ACD is closely similar to that of cABC. (B) The three registers of the ABC dimer observed by X-ray crystallography: AP_I (PDB ID code: 3L1G), AP_II (PDB ID code: 2WJ7), and AP_III (PDB ID code: 4M5S). Red lines indicate the vector between α-carbons of residues E117 on the two monomers, which is located on a twofold axis in AP_II. The distance between two modeled cysteines at position 117 is close enough for disulfide bond formation only in AP_II (9.13 Å between the two thiols in AP_I, 0.93 Å in AP_II, and 6.5 Å in AP_III). (C) ¹H-¹⁵N-HSQC spectra of cABC (blue) and cABC_E117C (orange) acquired under identical oxidizing solution conditions. Overlap of peaks is represented by purple. Peaks with significant shifts in the mutant are labeled on the plot. Dotted lines indicate peak movement in the mutant compared with cABC, and asterisks indicate unassigned peaks. The spectra overlay very well, indicating that the fold of the two proteins is very similar. (D) A heat map of CSP projected onto the structure of cABC (largest CSP, red; lowest, white) reveals that the most significant changes in chemical shift are observed near C117. (E) IM measurements of the cABC (blue) and cABC_E117C (orange) 7+ charge state (Fig. 4 E and F) under oxidizing conditions reveal very similar CCS distributions. Dashed lines indicate anticipated CCS values of AP_I, AP_II, and AP_III. Because the CCS distribution of cABC matches that of cABC_E117C, which is fixed as an AP_II dimer, we can infer that cABC preferentially populates the AP_II register.

Fig. 4.

Quaternary dynamics of cABC and cABC_E117C. (A) Nanoelectrospray MS of cABC reveals two charge-state distributions corresponding to an equilibrium of monomers (triangles) and dimers (squares). (B) A titration series of cABC reveals an increase in the abundance of dimer (dark blue) relative to monomer (light blue) as the concentration is increased (2, 4, 8, 16, and 32 μM, front to back). Spectra are normalized so that the most intense peak in each spectrum is equal to 100%. The shaded area indicates the 7+ charge state of the dimer. (C) A mass spectrum of cABC_E117C, obtained under the same conditions as for cABC in A, reveals the exclusive presence of dimers because of disulfide bond formation. (D) In the presence of reductant cABC_E117C reverts to a concentration-dependent equilibrium of monomers (orange) and dimers (brown) (concentrations are as in B) (E) A mass spectrum of unlabeled cABC mixed with ¹³C-labeled cABC, acquired as soon as possible, and focusing solely on the 7+ charge state (shaded in B). Both homo- and heterodimers are observed, indicating rapid subunit exchange. (F) An experiment equivalent to that in E carried out with cABC_E117C under reducing conditions demonstrates fast subunit exchange as observed for cABC.

Fig. 5.

In vitro chaperone activity of ACDs. (A) Dose-dependent inhibition of reduction-induced α-lac aggregation by cABC. Molar ratios are indicated as cABC:α-lac. Even at substoichiometric quantities of cABC, significant chaperone activity is observed. (B) Comparing different constructs of ABC at a molar ratio of 1:5 (chaperone:α-lac) reveals cABC to be equivalently potent to the full-length protein. cABC_E117C is slightly more effective than cABC, likely because of altered surface properties around the introduced cysteine (Fig. S3). (C) Assaying κ-casein fibrillation under reducing (Upper) and oxidizing (Lower) conditions in the presence of our ABC constructs at a chaperone:κ-casein molar ratio of 1:2 demonstrates that they are capable of slowing aggregation. cABC is less efficient than the full-length protein in both conditions, whereas cABC_E117C is more effective than cABC, mirroring the data in B. (D) The ABC constructs also are potent inhibitors of Aβ_1–42 fibrillation at a chaperone:Aβ_1–42 molar ratio of 1:20. Under reducing conditions (Upper), all three constructs perform equivalently, but under oxidizing conditions (Lower), cABC_E117C is more effective than cABC. This result suggests that locking into an AP_II dimer improves chaperone action in this assay. In all cases (A–D), a representative aggregation assay is shown as well as the percentage protection. Error bars correspond to mean ± SEM, with n = 3. *P < 0.05, **P < 0.01. (E) Cell-viability assays upon the addition of Aβ_1–42 to HEK293, HeLa, and PC12 cells. Aβ_1–42 on its own (red) decreases viability to 40%, but cABC (dark blue), cABC_E117C (brown), and buffer (gray) have no effect. Aβ_1–42 incubated in the presence of increasing amounts of cABC (blue) and cABC_E117C (orange) is less toxic than in the absence of chaperone. Molar ratios indicated are chaperone:Aβ_1–42, with an Aβ_1–42 concentration of 0.5 μM in all experiments (except the controls without Aβ_1–42). In all cases chaperone protection is clearly dependent on concentration. Error bars correspond to mean ± SEM, with n = 4. *P < 0.05.