Binding of Euplotes octocarinatus centrin to peptide from xeroderma pigmentosum group C protein (XPC)

Abstract

Centrins are Ca²⁺-binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. To probe whether Euplotes octocarinatus centrin (EoCen) could replace human centrin 2 (HsCen2), herein, we chose a 22-residue peptide (K842–R863) from the human xeroderma pigmentosum group C protein (XPC), a dominant component of the nuclear excision repair (NER) pathway, and investigated the detailed structural and energetic characterization of the interaction with EoCen using spectrophotometric methods, native PAGE and isothermal titration calorimetry (ITC). Fluorescence and UV difference spectroscopy revealed that the well-conserved tryptophan residue was buried in the hydrophobic pocket exposed by the C-terminal domain of EoCen. The native PAGE indicated that a new band appeared corresponding to a complex which was exclusively mediated by C-terminal domain of EoCen. Circular dichroism (CD) showed that peptide underwent a random coil-to-helix structural transition upon binding to the centrin, and ITC suggested centrin–peptide interactions were driven by an enthalpic contribution, which compensated for the unfavorable (negative) entropy term. Also, the affinity reduced by a factor of 4.67 compared with HsCen2, mainly due to V108 of the EoCen substitution for L112 of HsCen2.