N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity

Abstract

The aggregation of amyloid-β (Aβ) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease. Molecules binding with high affinity and selectivity to Aβ-peptides are important tools for investigating the aggregation process. An Aβ-binding Affibody molecule, ZAβ3 , has earlier been selected by phage display and shown to bind Aβ(1-40) with nanomolar affinity and to inhibit Aβ-peptide aggregation. In this study, we create truncated functional versions of the ZAβ3 Affibody molecule better suited for chemical synthesis production. Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges. The N-terminally truncated Affibody molecules ZAβ3 (12-58), ZAβ3 (15-58), and ZAβ3 (18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule ZAβ3 (1-58). Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, ZAβ3 (18-58), exhibited complete loss of binding to the Aβ(1-40)-peptide, while the ZAβ3 (12-58) and ZAβ3 (15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the Aβ(1-40)-peptide compared to the full-length Affibody molecule. Nuclear magnetic resonance spectroscopy showed that the structure of Aβ(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the Aβ(1-40)-peptide in complex with the full-length ZAβ3 Affibody molecule. This indicates that the N-terminally truncated Affibody molecules ZAβ3 (12-58) and ZAβ3 (15-58) are highly promising for further engineering and future use as binding agents to monomeric Aβ(1-40).

Figure 1

The amino acid sequences of the Aβ(1–40) peptide (A) and the Z_Aβ3(1–58) Affibody molecule (B). The arrows indicate the sites of truncation giving rise to the six different variants produced by solid phase peptide synthesis (SPPS). Note that the Z_Aβ3(1–58) sequence has an Asp²Glu substitution compared to the previously published Z_Aβ3 Affibody molecule. Block arrows indicate β-strands, while the cylinders indicate α-helical structure, as determined by the structure analysis of the complex done by Hoyer et al. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

CD spectra of titrations of Aβ(1–40) onto the four Affibody homodimers Z_Aβ3(1–58) (A), Z_Aβ3(12–58) (B), Z_Aβ3(15–58) (C), and Z_Aβ3(18–58) (D). Measurements were conducted at 25°C in 10 mM Na-phosphate buffer, pH 7.4, with an Affibody dimer concentration of 10 μM. Black line–1:0 Affibody dimer/Aβ(1–40) ratio; Blue line–1:0.2 ratio; Red line–1:0.4 ratio; Green line–1:0.6 ratio; Yellow line–1:0.8 ratio; Purple line–1:1 ratio.

Figure 3

Thermal melting profiles from 5 to 94 °C for different Affibody homodimers recorded with CD spectroscopy at 220 nm. Affibody dimer concentrations were 12 μM in 10 mM Na-phosphate buffer at pH 7.4. (A) Z_Aβ3(1–58); (B) Z_Aβ3(12–58); (C) Z_Aβ3(15–58); (D) Z_Aβ3(18–58). Solid profiles are for Affibody dimers alone; dashed profiles are for Affibody dimers in 1:1 complex with the Aβ(1–40) peptide.

Figure 4

SPR sensorgram showing the responses when different Affibody homodimers were injected over a chip surface to which Aβ(1–40) peptides were linked via a streptavidin/biotin system. Concentrations: Z_Aβ3(18–58) 200 nM; Z_Aβ3(15–58) 20 nM; Z_Aβ3(12–58) 20 nM; Z_Aβ3(1–58) 20 nM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

¹H¹⁵N-HSQC spectra of 100 μM ¹⁵N-labeled Aβ(1–40) at 25°C in 10 mM Na-phosphate buffer, pH 7.4, either alone (A) or in 1:1 complex with the unlabeled Affibody homodimers Z_Aβ3(18–58) (B), Z_Aβ3(15–58) (C), and Z_Aβ3(12–58) (D).

Figure 6

Assignments of ¹H¹⁵N-HSQC spectra of 250 μM ¹⁵N-labeled Aβ(1–40) at 25°C in 10 mM Na-phosphate buffer, pH 7.4. (A) Aβ(1–40) alone; (B) Aβ(1–40) in 1:1 complex with the unlabeled Affibody homodimer Z_Aβ3(12–58).

Figure 7

Chemical shift differences (Δδ = (((Δδ_N/5)² + (Δδ_H)²)/2)^1/2) between HSQC spectra of ¹⁵N-labeled Aβ(1–40) in its free form and bound to the Affibody homodimers Z_Aβ3(12–58) (A) and Z_Aβ3(15–58) (B).