Binding and inhibition of copper ions to RecA inteins from Mycobacterium tuberculosis.
Protein splicing is a unique post-translational process in which an intein excises itself from a precursor with the concomitant ligation of flanking sequences. The binding of zinc to intein inhibits protein splicing reversibly and EDTA relieves the inhibition. Copper was found to inhibit protein trans splicing; however, the recovery of intein splicing required both EDTA and TCEP, suggesting a different inhibition mechanism for copper compared to zinc. In this work, we have investigated the binding properties and inhibition effects of copper ions on the RecA intein from Mycobacterium tuberculosis. Both Cu(+) and Cu(2+) exhibited high binding affinity to inteins, while different binding sites were identified. Cu(2+) coordinates to Cys1, the key residue involved in the mechanism of protein splicing, however, Cu(+) does not coordinate to cysteine. An in vitro inhibition assay indicated that monovalent Cu(+) demonstrates reversible inhibition to protein splicing, and the inhibitory efficiency is comparable to Zn(2+). Redox reaction between Cu(2+) and cysteine in inteins were observed and the rate constants were determined. The results suggested a dual role for Cu(2+) in the inhibition of intein splicing: strong coordination of Cu(2+) to key residues (including Cys1) in the intein, and subsequent oxidation of Cys1, the residue required for the N-->S acyl shift step in protein splicing. A kinetic study suggested that the coordination could be the major cause of inhibition effect of Cu(2+) initially, whereas the redox reaction could play an additional role in inhibition at a later stage.
Zhang
Liyun
L
Department of Chemistry, CAS Key Laboratory of Soft Matter Chemistry, University of Science and Technology of China, 230026 Hefei, Anhui, China.
Xiao
Ning
N
Pan
Yan
Y
Zheng
Yuchuan
Y
Pan
Zhiyun
Z
Luo
Zhaofeng
Z
Xu
Xiaolong
X
Liu
Yangzhong
Y
D001665:Binding Sites; D003300:Copper / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D047668:Inteins; D007477:Ions / Q000737:chemistry*; D008026:Ligation; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D019154:Protein Splicing; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D007477:Ions; D003300:Copper; D004492:Edetic Acid; D011938:Rec A Recombinases; D015032:Zinc