Nickel(ii)-promoted specific hydrolysis of zinc finger proteinsElectronic supplementary information (ESI) available. See DOI: 10.1039/c8mt00098k

• Main Authors: Belczyk-Ciesielska, Agnieszka, Csipak, Brigitta, Hajdu, Bálint, Sparavier, Aleksandra, Asaka, Masamitsu N, Nagata, Kyosuke, Gyurcsik, Béla, Bal, Wojciech
• Format: Journal Article
• Published: 15-08-2018
• ISSN: 1756-5901; 1756-591X
• DOI: 10.1039/c8mt00098k

In this work we demonstrate that the previously described reaction of sequence specific Ni(
ii
)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys
2
His
2
zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(
ii
) ions. It results in loss of the Zn(
ii
) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(
ii
)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(
ii
)-susceptible sequence between the edited zinc finger and an affinity tag allows for removal of the latter sequence by Ni(
ii
) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(
ii
) ions. Mass spectra revealed that a Ni(
ii
) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(
ii
)-dependent protein hydrolysis is influenced by the Ni(
ii
) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.
The (S/T)XH sequence in Cys
2
His
2
zinc fingers can be hydrolytically cleaved by Ni(
ii
) ions. This reaction can be applied for purification, inhibition or activation of designed zinc finger fusion proteins.