Abstract
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 Cys2His2 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.
Highlights
We reported on Ni(II)-induced peptide bond hydrolysis that occurs at the N-terminal side of Ser or Thr residues in peptides,[26,27,28,29,30,31,32,33,34,35,36] designed recombinant proteins[37,38] and flexible regions of native proteins[37,39] bearing X-(Ser/Thr)X-His-X sequences (X being any amino acid except for Cys or Pro before or after Ser/Thr)
The P-1MEY zinc finger (ZF) protein sequence is based on the consensus sequence CP-1 derived by Berg et al.,[46] and confirmed by a recent study,[47] with the exception of the amino acids responsible for DNA recognition
Short extensions at both termini of the P-1MEY protein sequence can be derived from the consensus sequence as they are found in the Zinc Finger Tools program used for protein redesign.[13]
Summary
We reported on Ni(II)-induced peptide bond hydrolysis that occurs at the N-terminal side of Ser or Thr residues in peptides,[26,27,28,29,30,31,32,33,34,35,36] designed recombinant proteins[37,38] and flexible regions of native proteins[37,39] bearing X-(Ser/Thr)X-His-X sequences (X being any amino acid except for Cys or Pro before or after Ser/Thr). Various human transcription factors contain potential Ni(II) cleavage sites within their Cys2His[2] ZF units. The 27 amino acid long peptide, representing the 3rd ZF unit of the Sp1 human transcription factor was shown to be cleaved by Ni(II) ions even though the His residue crucial for the reaction was initially complexed to the structural Zn(II) ion.[32] The Zn(II) binding site and the structure of the ZF unit was abolished in the reaction. Occurring Ni(II)-cleavage sites, which are present in nearly 25% of human Cys2His[2] ZF sequences[32] can be considered potentially susceptible to hydrolysis upon the exposure to Ni(II) ions naturally present in human tissues.[41] Nickel is absorbed through the diet, skin, lungs and taken up by cells.[42,43] By analogy with the hydrolytic cleavage of histone H2A observed in Ni(II)-exposed cell cultures, endogenous nickel present in human cells may damage ZFs as part of its toxicity
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