Nces in RMSF values have been proved to be mechanistically relevant for Fdx [46].Figure 5. Analysis of BsNTD, TmNTD and PMS1 monomers residues mobility using structure based models (SBM). Root Mean Squeare Fluctuation (RMSF) difference between apo and ATPbound (full black line) and ADP and ATP-bound monomer (dotted line) for; A) BsNTD B) TmNTD and C) yeast PMS1 were calculated from SBM MD simulations. Dimerization interface is indicated with black horizontal bars. ATP lid residues are marked with grey shadow. doi:10.1371/journal.pone.0069907.gInfluence of Nucleotide Binding on PaNTD-PaCTD InteractionSince theoretical results of SBM MD and mixed-solvent MD indicate that PaNTD protein-protein interactions could be nucleotide modulated, we tested PaNTD-PaCTD binding capacity in absence or in presence of ATP or ADP by far western (Figure 9 and Figure S7). PaCTD spotted onto nitrocellulose membranes was incubated in buffer with His-tag PaNTD in absence or in presence of ATP or ADP, and PaCTD-PaNTD complexes were revealed and quantified using a His-tag antibody.the server DNABindR [44] for prediction of protein-DNA interaction sites was used to predict PaNTD residues involved in DNA contact (Figure S6). This server is trained to predict whether a given amino acid residue is a DNA-binding residue based on itsPLOS ONE | www.plosone.orgMutL N-Terminal Domain InterfacesFigure 6. Determination of PaNTD oligomeric state using gel filtration chromatography. A typical elution profile is shown: (A) PaNTD and (B) EcNTD proteins (22 mM) nucleotide free (full black line), bound to ADP (dotted line) or bound to ATP (full grey line) were analyzed on a Superose 12 column as described in Matherial and Methods.Nipocalimab Arrow heads indicate the elution positions of MW standards (= BSA 66 kDa; .FGF-8b Protein, Human/Mouse BSA 45 kDa).PMID:26760947 doi:10.1371/journal.pone.0069907.gWhereas no difference where observed in PaNTD-PaCTD binding when PaNTD was incubated with buffer or ATP, an increased interaction was observed when incubated with ADP (Figure 9). PaNTD-BSA binding was used as a negative control whereas His-PaNTD was directly spotted onto membranes as a positive control (Figure S7). Far western results were statistically analyzed using an analysis of variance (ANOVA) followed by a Tuckey HSD test. These tests allowed us to determine that mean NTD-CTD interaction in presence of ADP was significantly higher than NTD-CTD interaction in presence of ATP or in absence of nucleotide (buffer) with a p value of 0.05 and 0.01, respectively. Also, no differences in NTD-CTD interaction were found for ATP vs. buffer incubation. These results are consistent with theoretical data mentioned before, and indicate that binding of adenosines nucleotides coulddifferentially affect the exposure of protein-protein interaction sites. This is in agreement with the assignment of a main role of nucleotide binding in PaMutL endonuclease activity modulation [5,7,9]. Nevertheless, further experiments would reinforce this observation and help to elucidate adenine nucleotides role in NTD-CTD interaction.DiscussionSeveral MutL homologues from organisms lacking MutH have been shown to possess an endonuclease activity that requires the integrity of a metal-binding motif located in MutL C-terminal domain [3,9,10]. In human and yeast MutLa, endonuclease activity has been proved to be nick-directed and mismatchPLOS ONE | www.plosone.orgMutL N-Terminal Domain InterfacesFigure 8. Effect of adenine nucleotide binding on PaNTD dimers. SBM si.