Study On The Role Of The First Asp Residues Of EF-loops In Ciliate Euplotes Octocarinatus Centrin

Centrin is an acidic, low molecular weight (20 kDa) protein that belongs to the EF-hand superfamily of calcium binding proteins. This calcium binding protein (centrin) was first identified as a major component of the fibers that link the nucleus to the flagellar apparatus in flagellated unicells, and later, it was shown to be a ubiquitous component of centrioles, centrosomes, and mitotic spindle poles. Genetic studies have shown that centrin is required for cell cycle-dependent duplication and segregation of the MTOC.Ciliate Euplotes octocarinatus centrin (EoCen) is a protein of 168 residues. Centrin is a highly conserved member of calcium-binding proteins which contain four helix-loop-helix domains, the so-called EF-hands, which may each bind one Ca2+. For centrin, the first amino acid residues (aspartic acids) of four EF-hand loops are also highly conserved. Thus, the Asp in the beginning of the loops would be expected to be important to the proper metal-binding characteristic and function of centrin. In this work, EoCen and the mutants (D37K, D73K, D110K and D146K) were constructed, recombinated, expressed, and purificated. Asp37, Asp73, Asp110 and Asp 146, the first amino acids of the EF-loops of centrin, respectively, were mutated to lysine (Lys). Then the role of the four conversed aspartic acid in metal-binding characteristic of EoCen were studied as follow.Firstly, using fluorescence spectra, the metal-binding characteristic of four EF-loops of EoCen was investigated in physical condition. The results of Tb3+ fluorescence sensitivity indicated that the ability of Tb3+ binding was abolished by the mutation of the first aspartic acid residue of each EF-loop, except for loop II (Asp73). Based on fluorescence titration curves of Lu2-D37K, the conditional binding constants of EoCen loopⅡquantitatively to be KⅡ= (1.61±0.04)×105 L·mol-1 and KⅡ= (3.52±0.08)×102 L·mol-1 with Tb3+ and Ca2+ were reckoned, respectively. The order of metal-binding affinity of these four sites in EoCen is obtained, namely IV>Ⅲ>Ⅰ>Ⅱ. Resonance light scattering experiment shows that the different degree of self-assembly is found among the mutants, namely N-terminal domain plays a critical role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV), there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, TNS binding experiment, ionic strength experiment and pH control experiment show that in the process of EoCen self-assembly, molecular interactions are mediated by hydrophobic forces, and the electrostatic interaction exists truly. The order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen induced by Tb3+ is Asp37> Asp 146> Asp110> Asp73, and Asp37 plays the most important role in the self-assembly of EoCen.Far-UV circular dichroism spectra of the single mutants are highly similar to that of wild type EoCen, suggesting that the mutation of any aspartic acid does not disrupt the secondary structure of wild-type EoCen. TNS was applied to detect the conformational change of WT-EoCen and all the mutants in the absence or presence of Ca2+ and Tb3+ as the hydrophobic probe. The hydrophobic surfaces of mutants were different, the order of the four conserved aspartic acid residues for contributing on the proper conformation of EoCen is Asp37>Asp146>Asp110>Asp73.Molecular modeling indicated that the ability of metal-binding of EF-loopⅠ,ⅢandⅣwere lost by the mutation of Asp37, Asp110 and Asp146, the ability of metal-binding of EF-loopⅡwas not lost, which is consistent with experiment results.