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-β 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 Cys955 or His960 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.
Highlights
Really interesting new gene (RING) finger is a characteristic protein sequence motif that was first identified in the protein product of the human gene RING1—Really Interesting New Gene 1—which is located proximal to the major histocompatibility region on chromosome 6 [1]
RING finger motifs are further subdivided, depending on whether a cysteine or histidine residue is found at Cys/His5 within the motif
The C955R mutant exhibits expression yield sufficient for recombinant protein isolation and the protocol applied was based on that used for the wt of Arkadia RING-H2 finger domain [21]
Summary
RING finger is a characteristic protein sequence motif that was first identified in the protein product of the human gene RING1—Really Interesting New Gene 1—which is located proximal to the major histocompatibility region on chromosome 6 [1]. Substitution of various native amino acids, especially the zinc-binding residues, resulted in the significant loss of ubiquitination capacity, as shown in various E3 ligases such as BRCA1, HDM2 and many other RING finger domaincontaining proteins. Substitution of zinc-binding amino acids with residues by atom-donors with lower affinity to zinc metal ion, results in a collapsed RING finger structure or in a conformational alteration that prevents RING from interacting with E2.
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