Modulation Of XPC Peptide On The Properties Of Euplotes Octocarinatus Centrin

Centrins are small?MW²0 kD?,acid,Ca²⁺-binding proteins that belong to the EF-hand calmodulin superfamily.Centrin is organized into two independent global domain tethered by a central linker,each domain consisting of a pair of EF-hand motifs of helix-loop-helix that can potentially bind two Ca²⁺ions.The location of the centrin is not limited to centrosome,which are structurally and functionally similar to basal bodies in ciliated and flagellated cells,the spindle pole bodies in yeast,throughout the eukaryotic kingdom and centrins are distributed in other cell compartments such as in the perimembrane space or nucleus.It is required for proper cell division and plays significant roles in the contraction of fibers based-centrin among eukaryotic cells.Ciliate Euplotes octocarinatus centrin?EoCen?was first reported by our group?GenBank accession number:Y18899?,which was cloned from unicellular eukaryotic organism Euplotes octocarinatus.EoCen consists of168 residues,wherein there are 4 tyrosines,10 phenyalanines and no cysteine.From sequence analysis,it shares approximately 50%sequence identity with the most studied EF-hand protein CaM,and shares about 60,62 and 66%sequence identity with human centrin 1?HsCen1?,human cntrin2?HsCen2?and human centrin 3?HsCen3?,respectively.A 22-residue peptide?K842-R863?from xeroderma pigmentosum group C protein?XPC?was used to investigate the peptide recognition of EoCen.Based on the peptide binding properties,we elucidated the relationship between target peptide recognition and Tb³⁺binding/self-assembly of EoCen in vitro.Expression and purification of the EoCen and its fragments?N-EoCen,C-EoCen,?23EoCen,?23N-EoCen,G115W and G151W?were carried out according to canonical protocols.The interaction between EoCen and XPC peptide was characterized by fluorescence spectroscopy,UV spectroscopy,circular dichroism?CD?,isothermal titration calorimetry?ITC?and nondenaturation polyacrylamide gel electrophoresis in physiological conditions.Fluorescence and UV difference spectra revealed that the well-conserved and anchoring tryptophan residue in XPC peptide transferred from the polar water solvent to the apolar hydrophobic package created by two EF-hand motifs of C-terminal domain.The association constant between XPC peptide and EoCen was 5.88×10⁶ M^-1,and 4.55×10⁶M^-11 for C-EoCen.Spectrofluorimetric titration represented the formation of a 1:1 complex,suggesting there is only one XPC binding site in centrin and the N-terminal domain was not engaged in peptide binding.CD analysis suggested that the peptide underwent a random coil-to-helix transition upon binding to the protein.The new complex was visualized by native PAGE.The affinity constants were 1.67×10⁶ M^-11 and 0.46×10⁶ M^-11 for EoCen and C-EoCen,respectively,by ITC experiments and the peptide recognition was exothermic reaction.Indeed,the free energy of the peptide binding to C-EoCen contributed 92%of that of the integral protein.The interaction of two mutants?G115W and G151W?of EoCen with XPC confirmed that XPC peptide could not touch with the residues in loop of C-terminal domain,and only touch with the hydrophobic package created by EF-hand motifs.The predominant force was hydrophobic interaction and the binding mode was extended mode,which was similar to HsCen2-XPC.Ca²⁺is centrin's native cation which is silent in spectroscopic,electrical and magnetic properties.Due to the similar coordination chemical properties and ionic radii of calcium ion and lanthanide ion,rare earth ions,nonbiogenic metal species,can be widely used to probe the effect of metal ions on the peptide binding and the effect of peptide recognition on the Tb³⁺binding of EoCen.In the presence of metal ions(Ca²⁺or Tb³⁺),the N-terminal domain of EoCen was still not involved in the interaction with XPC peptide.The affinity obtained from spectrofluorimetric titrations and ITC manifested that Ca²⁺and Tb³⁺could enhance the peptide binding weakly.The affinity order of different forms protein both were Tb³⁺>Ca²⁺>apo,which was in perfectly agreement with the degree of exposure of hydrophobic interface and conformation change induced by metal ions.The peptide recognition was still exothermic reaction in the presence of metal ions,and was driven by enthalpy and entropy.Native PAGE and RLS analysis showed that the peptide recognition resulted in the dissociation of the aggregates.Complex came into being by monomer-peptide rather than by multimer-peptide form.XPC peptide slightly enhanced the affinity of the C-terminal domain of EoCen to Tb³⁺,while decreased dramatically the affinity of Tb³⁺to the N-terminal domain of EoCen,about 225 times.A truncated form of EoCen,lacking the first 23 residues??23EoCen?,consists of the unbroken C-terminal domain which is the critical anchoring region of XPC,so showed conspicuous peptide binding.Native PAGE,fluorescence spectroscopy and ITC indicated the character of peptide binding was similar to EoCen.?23EoCen can not aggregate even in the presence of metal ions.RLS signal was larger than that of?23EoCen in the presence of XPC peptide.EoCen can aggregate in the presence of metal ions,especially Tb³⁺binding to the N-terminal domain of EoCen.Since the addition of XPC peptide affected the Tb³⁺binding capacity of the N-terminal domain of EoCen severely,the self-assembly of EoCen was inhibited strongly.There was four metal ions binding sites in?23EoCen,and XPC recognition had no effect on Tb³⁺binding to?23EoCen,which was similar to EoCen.RLS induced by Tb³⁺,native PAGE and chemical cross-linking analysis indicated that the critical role played by the N-terminal extension in the self-assembly of EoCen.Only N-EoCen and EoCen can aggregate while?23N-EoCen,?23EoCen and C-EoCen can not aggregate.Chemical cross-linking analysis demonstrated the N-terminal extension?N-23?could be cross-linked with?23N-EoCen,?23EoCen and C-EoCen.Contrary to the reaction heat of dissociation of aggregates,the binding of N-23 to?23EoCen was exothermic reaction,indicating the self-assembly of EoCen came true via the interaction of N-23 with?23EoCen.The affinity between N-23 and?23EoCen reduced by a factor of 42 compared with the affinity between?23EoCen and XPC,suggesting target peptide binding was stronger than the self-assembly in vitro.Fluorescence intensity of the mixture consisting of EoCen cross-linked by glutaraldehyde and XPC decreased by 63%.The self-assembly of EoCen induced by Tb³⁺was inhibited in the presence of XPC peptide,and the self-assembly of N-EoCen was not impacted by peptide.Native PAGE confirmed that peptide binding inhibited the self-assembly induced by Tb³⁺,because Tb³⁺dissociated from the N-terminal domain of Tb³⁺saturated EoCen,which was favorable for the self-assembly of EoCen.In summary,the binding of metal ions benefited to peptide recognition and self-assembly of EoCen;the removal of metal ions led to the dissociation of the aggregates.Peptide binding decreased the affinity of Tb³⁺to the N-terminal domain of EoCen,so the self-assembly induced by Tb³⁺was inhibited.In addition,peptide binding resulted in the dissociation of EoCen aggregates;Peptide binding of EoCen cross-linked by glutaraldehyde decreased remarkably.EoCen-XPC peptide Complex came into being by monomer-peptide rather than by multimer-peptide form.XPC peptide was used to probe the stability of EoCen-XPC induced by urea,the unfolding of EoCen-XPC was also a two-transition,three-state process.The first transition corresponded to the unfolding of the N-terminal domain and the second transition was attributed to the unfolding of the C-terminal domain.Metal ions(Ca²⁺and Tb³⁺)stabilized complex,and the extent of stability of Ca²⁺and Tb³⁺was almost the same.In the same experimentalconditions,Gibbsfreeenergiesofunfoldingof?23EoCen-XPC and EoCen-XPC were close,indicating that N-23 fragment had no effect on the peptide recognition and the stability of EoCen.The unfolding curve was obtained from the spectral change of EoCen-XPC complex,so Gibbs free energy of protein induced by urea was larger than that induced by GdnHCl.The unfolding of G115W?mutant of EoCen?induced by GdnHCl was a two-transition,three-state process.The first transition belonged to the dissociation of the EoCen aggregates,and the second transition was assigned to the unfolding of protein.Tb³⁺could enhance the stability of protein,wherein Tb³⁺situated at high affinity sites?site III and IV?had negligible impact on the stability of protein,and Tb³⁺located at low affinity sites?site I and II?yielded considerable influence on protein stability.