Modulation of XPC peptide on binding Tb³⁺toEuplotes octocarinatuscentrin

Abstract

Centrins are Ca-binding proteins found throughout eukaryotic organisms. Xeroderma pigmentosum group C protein (XPC), a dominant component of the nuclear excision repair (NER) pathway, is a critical target protein of centrins. A 22-residue peptide (K842-R863) from XPC was used to investigate the effect of metal ions (Ca and Tb) on the peptide binding of Euplotes octocarinatus centrin (EoCen) by isothermal titration calorimetry (ITC) and fluorescence spectroscopy. ITC and tryptophan spectrofluorimetric titrations revealed that metal ions (Ca and Tb) could enhance the affinity between EoCen and the XPC peptide, and the enhanced effects were closely related to the ion potential of metal ions. Since the ion potential of Tb (e/r = 0.0325) is larger than that of Ca (e/r = 0.0202), the conformational change in the protein induced by Tb is larger than that induced by Ca, and the enhanced affinity of Tb is stronger than that of Ca. This interaction was driven by enthalpy in the presence of EDTA and enthalpy and entropy in the presence of Ca or Tb. Similar to that observed in the presence of EDTA, the N-terminal domain did not participate in the interaction with the XPC peptide even in the presence of metal ions. Resonance light scattering (RLS) and the band shift in native polyacrylamide gel electrophoresis (PAGE) suggested that peptide binding resulted in the dissociation of EoCen aggregates and complex formation via the monomer-peptide form. Tb-Sensitized emission suggested that peptide binding in turn also had an impact on the Tb binding of the protein: the C-terminal domain was slightly strengthened and the N-terminal domain was weakened about 225 fold. RLS and native PAGE indicated that the self-assembly induced by Tb binding to the N-terminal domain of EoCen was inhibited in the presence of the XPC peptide. This study elucidates the molecular mechanism of EoCen function in the cellular context.

The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii

“…Accordingly, the hydrodynamic radius of TgCEN1 particles decreased upon addition of P17-XPC (from 570 ± 5 nm to 307 ± 1 nm) ( Figure 7B ). These results agree with previous observations on the centrins from S. cerevisiae (Cdc31p), S. dubia (SdCEN), HsCEN1 and HsCEN2 for which the presence of the binding peptide Kar1p (centrosomal target protein) caused a strong reduction in the formation of multimeric structures [ 14 ] and with the observation that addition of XPC peptide to centrin from Euplotes octocarinatus (EoCEN) resulted in the dissociation of protein aggregates [ 43 ].…”

“…7B). These results agree with previous observations on the centrins from S. cerevisiae (Cdc31p), S. dubia (SdCen), HsCEN1 and HsCEN2 for which the presence of the binding peptide Kar1p (centrosomal target protein) caused a strong reduction in the formation of multimeric structures [14] and with the observation that addition of XPC peptide to centrin from Euplotes octocarinatus (EoCEN) resulted in the dissociation of protein aggregates [43]. Of note, addition of P17-XPC to N-TgCEN1 had no effect on the association properties of this domain.…”

Phosphorylation promotes the endonuclease-like activity of human centrin 2

“…Metal ions combine with appropriate proteins to form metalloproteins that drive different biological functions in the body. 42 In order to visually detect the interaction between Tb 3+ and protein, FRET from the tyrosine residue on protein to bound Tb 3+ was measured. When Tb 3+ is added to HsCen2, the nonradiative energy transfer from tyrosine residue on HsCen2 to the bound Tb 3+ resulted in the uorescence sensitization of four weak characteristic peaks of Tb 3+ at 490, 545, 585, and 620 nm (Fig.…”

“…40,41 Our previous research proves that the interaction between HsCen2 and XPC peptide is double-regulated by phosphorylation and Ca 2+ . 34 As terbium ions (Tb 3+ ) have similar chemical hardness, ion radius, coordination number, and electrostatic coordination characteristics to Ca 2+ , 42,43 Tb 3+ has been widely used as a uorescent probe to study Ca 2+ -binding proteins.…”

“…As terbium ions (Tb 3+ ) have similar chemical hardness, ion radius, coordination number, and electrostatic coordination characteristics to Ca 2+ , 42,43 Tb 3+ has been widely used as a fluorescent probe to study Ca 2+ -binding proteins.…”