estriol In silico analysis revealed structural similarities
In silico analysis revealed structural similarities between the CpMutY and the EcMutY which share typical domains and structural motifs (Fig. 5), common to a wide range of structurally related DNA repair enzymes. These include an endonuclease III domain, involved in excision of damaged pyrimidines, and a MutY DNA glycosylase domain, an important enzyme of the GO system, which removes estriol from 8-oxoG:A mismatches (Nagorska et al., 2012, Robles et al., 2011, Williams and David, 2000). HhH (helix-hairpin-helix), the DNA binding motif predicted for CpMutY, is found in enzymes that bind DNA with no sequence specificity, through interactions mediated by hydrogen bonds with DNA phosphate groups (Doherty et al., 1996, Michaels et al., 1990, Shao and Grishin, 2000). The iron-sulfur cluster structural motif, referred to as the 4Fe-4S cluster loop (FCL or FES) is composed of four cysteine residues in a specific pattern (C-X6-C-X2-C-X5-C) and spatial conformation, constituting the region to which the [4Fe-4S] group is bound (Doherty et al., 1996). The basic residues in this motif are preserved in CpMutY and are arranged so as to interact with the phosphate groups of DNA. It has been suggested that this motif is closely involved in the process of adenine excision by E. coli MutY, and thus the [4Fe-4S] cluster is required for enzyme binding to DNA and is essential for MutY adenine glycosylase activity (Boal et al., 2007, Chepanoske et al., 2004, Eberle et al., 2015, Oliveira et al., 2014, Porello et al., 1998). The similarity observed between the CpMutY and MutY from E. coli, H. pylori, P. gingivalis and M. tuberculosis suggests they share important preserved functional regions known for their role in the 8-oxoG:A mismatch repair and in the prevention of spontaneous mutagenesis (Huang et al., 2006, Michaels and Miller, 1992, Robles et al., 2011). Conserved catalytic Glu37 and Asp138 residues (positions relative to E. coli MutY) (Fig. 5) are essential for maintaining glycosylase activity in MutY and were found in C. pseudotuberculosis (Glu55 and Asp156) (Guan et al., 1998, Wright et al., 1999). The Val45 residue present in E.coli is also conserved in CpMutY (Val63) and is involved in adenine-specific activity of E. coli MutY (Chang et al., 2009). Furthermore, the four cysteine residues preserved in the binding domain of the [4Fe-4S] iron-sulfur cluster, are important for the maintenance of enzymatic function, since directed mutations affect the repair capacity of E. coli MutY (Boal et al., 2007). Regarding CpMutY, all important residues for DNA-binding, damage recognition, and catalytic activity are maintained and provide evidence for its functional and structural conservation (Fig. 5, Fig. 6). The structural similarity between CpMutY and G. stearothermophilus MutY was observed in the three-dimensional structure constructed by comparative modeling. The structural organization of the central region of the constructed theoretical model (Fig. 7) closely resembles that found in the catalytic domain of MutY from G. stearothermophilus (Fig. 6) and Bacillus stearothermophilus (Fromme et al., 2004, Guan et al., 1998). The region is crucial for excision of the mismatched base, since it comprises the extra-helical pocket into which the mismatched adenine is inserted in order to be excised, extensively interacting with the DNA chain (Fromme et al., 2004, Lee and Verdine, 2009). The C-terminal region exhibits fewer conserved residues, as seen in the alignment of sequences. Although not essential for the catalytic activity of MutY adenine glycosylase, the C-terminal portion determines the specificity for the 8-oxoguanine lesion, since the absence of this domain reduces the enzyme's preference for the 8-oxoG:A substrate, favoring instead its affinity for A:G (Li et al., 2000). Additionally, the protein depicts an overall positive surface (Fig. 8), with a total charge of 7e, consistent with its function and characteristic of DNA-binding proteins. We then turned to assess the structural and functional effects of tyrosine-to-lysine and tyrosine-to-serine mutations at CpMutY position 138. To this end we have generated in silico mutated three-dimensional models of these proteins and molecular docking studies were carried out to point out the differences in DNA-binding capacities of the wild-type compared to both mutated proteins. As a result, we have observed a misplacement of the adenine base on the Y138S mutated structure, when compared with its wild-type counterpart. A difference of over 6Å was calculated when measuring the distance between the adenine and the residue on position 138 of CpMutY. The chosen molecular docking methodology, implemented in Haddock, defines ambiguous interactions restraints (AIRs) to drive the docking process (Cyril et al., 2003). An AIR is defined by the authors as an ambiguous distance between all interacting residues. Therefore, lower AIRs point to closer molecular interactions and more native-like protein-DNA complexes. The Y138S CpMutY-DNA complex has an AIR energetical component of over 70, which is >20 times greater than the same value for both the wild-type and Y138K protein-DNA complexes.