Metal Binding Properties of the N-Terminus of the Functional Amyloid Orb2

Abstract

The cytoplasmic polyadenylation element binding protein (CPEB) homologue Orb2 is a functional amyloid that plays a key regulatory role for long-term memory in Drosophila. Orb2 has a glutamine, histidine-rich (Q/H-rich) domain that resembles the Q/H-rich, metal binding domain of the Hpn-like protein (Hpnl) found in Helicobacter pylori. In the present study, we used chromatography and isothermal titration calorimetry (ITC) to show that the Q/H-rich domain of Orb2 binds Ni²⁺ and other transition metals ions with μM affinity. Using site directed mutagenesis, we show that several histidine residues are important for binding. In particular, the H61Y mutation, which was previously shown to affect the aggregation of Orb2 in cell culture, completely inhibited metal binding of Orb2. Finally, we used thioflavin T fluorescence and electron microscopy images to show that Ni²⁺ binding induces the aggregating of Orb2 into structures that are distinct from the amyloid fibrils formed in the absence of Ni²⁺. These data suggest that transition metal binding might be important for the function of Orb2 and potentially long-term memory in Drosophila.

Keywords: ITC; aggregation; amyloid; protein–metal interaction; thioflavin T fluorescence.

Figure 1

Domain structure of rare isoform Orb2A that is necessary to initiate fibril formation and the predominant isoform Orb2B highlighting the glutamine, histidine rich domain (Q/H), the glycine, serine rich region (G/S), the two RNA recognition motifs (RRM) and the zinc finger (Zn) motif. The sequence of the Q/H-rich domain is shown with all histidines in red and the hisitidines that were mutated in this study highlighted in yellow.

Figure 2

Orb2A87 binds several transition metal ions in the absence of a polyhistidine-tag. Coomassie stained SDS-PAGE showing the purification results of Orb2A87 using nitrilotriacetic acid (NTA) agarose charged with nickel, copper, cobalt, and zinc as well as metal-free NTA agarose. Orb2A87, unlike Orb2A88, has no polyhistidine-tag bound to all of these metals, but did not bind to metal-free NTA agarose.

Figure 3

Orb2A87 binds to Ni²⁺, Zn²⁺ and Cu²⁺ but not to Mg²⁺. Isothermal titration calorimetry (ITC) titration curves of Orb2A87 with the addition of Ni²⁺ (A); Zn²⁺ (B); Cu²⁺ (C); and Mg²⁺ (D). Both the heat compensation in the sample cell containing Orb2A87 as a function of time (top) and the resulting enthalpy changes as a function of molar addition of metal and the best fit to single binding site model (bottom) are shown.

Figure 4

Orb2A87 mutants H29A, H46A and H60A still bind to Ni²⁺ but only with half a binding site per monomer (A–C). ITC Ni²⁺ titration curves of Orb2A87 mutants, H29A (A); H46A (B); H60A (C). Both the heat compensation in the sample cell containing Orb2A87 as a function of time (top) and the resulting enthalpy changes as a function of molar addition of metal and the best fit to single binding site model (bottom) are shown; (D) H61Y does not bind to Ni-NTA resin. SDS-PAGE comparing the Ni-NTA pH 4.25 elution fraction of wild type (WT) Orb2A87 with Orb2A87 H61Y.

Figure 5

Mg²⁺ and Ca²⁺ do not change the structure of Orb2A87. Cu²⁺, Ni²⁺, and Zn²⁺ lead to a decrease in circular dichroism (CD) signal. CD spectra of Orb2A87 without metal and in the presence of 2.5 molar surplus of Cu²⁺, Ni²⁺, Zn²⁺, Ca²⁺, and Mg²⁺. The presence of Ca²⁺ and Mg²⁺ does not change the CD spectrum compatible with Orb2A87 in a predominantly random coil conformation. The addition of Cu²⁺, Ni²⁺, Zn²⁺ leads to a decay of CD signal with no indication of additional secondary structure.

Figure 6

Addition of Ni²⁺ induces rapid increase of thioflavin T (Tht) fluorescence. (A) Tht fluorescence kinetics of 17.64 μM Orb2A87 in the absence of metal (black) and in the presence of 2.5 molar excess of Ni²⁺ (cyan) and Mg²⁺ (pink). Buffer control with and without metals shows no fluorescence increase. The average and error of three technical repeats is shown; (B) Average of the Tht kinetics half times (T_1/2) calculated from three independent repeats of the Tht time courses shown in (A).

Figure 7

Addition of Ni²⁺ induces aggregation distinct from fibril formation. Electron micrographs of negatively stained aggregates of Orb2A87 collected 6 hours after the start of an aggregation study. Orb2A87 without metal and in the presence of a 2.5 molar excess of Mg²⁺ show typical bundled amyloid fibrils. Aggregates that formed in the presence of a 2.5 molar excess of Ni²⁺ look rather amorphous although high Tht fluorescence was observed in this case. Scale bars: 200 nm.