Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

Abstract

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF-beta signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues Cys(955) or His(960) to Arginine, through site-directed mutagenesis. The recombinant expression, in Escherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.

Figure 1

(a) The schematic structure of a RING finger domain showing the “cross-brace” arrangement of the two zinc binding sites; (b) The amino acid sequence of the RING finger domain of mouse Arkadia; (c) the mutation sites, C955R and H960R, in the sequence of Arkadia RING Finger domain.

Figure 2

The total cell lysate of wt (a), the mutant C955R (b), and H960R (c) at 37°C with ZnCl₂ at a final concentration 100 μM.

Figure 3

Calibration curves for the additions of zinc in (a) RING-H2 and (b) mutant RING (C955R) of Arkadia.

Figure 4

The 1D ¹H-NMR of amide proton resonances (7,5–10 ppm) and ¹H-¹⁵N HSQC of the mutant (a) and the native RING (b).