Ected as high positive DRMSF values. On the other hand, DRMSF of PaNTD with ATP or ADP bound states where quite similar (Figure 4B, dotted line). This is reflected as near zero DRMSF values, and indicates that these ADP and ATP-bound PaNTD behave similarly. Additionally, MD simulations of SBM were performed to analyze differences between NTD from MutL homologues with/ without endonuclease activity (Figure 5). The N-terminal domains of MutL homologues with endonuclease activity from Bacillus subtillis (BsNTD), Termotoga maritima (TmNTD) and PMS1 from Saccharomyces cerevisiae (yNTD) were analyzed. WebMod server [25] was used to model BsNTD and TmNTD 3D structure, since these proteins tertiary structures have not been determined experimentally. SBM MD simulations were carried out for the four proteins in the nucleotide free (apo) form or bound either to ADP or ATP. Differences in RMSF (DRMSF) between (i) the apo and ATP bound forms or (ii) ADP and ATP of each protein were determined (Figure 5). For BsNTD a model was constructed using EcNTD template (PDB:1B63A).3-Aminobenzamide BsNTD (Figure 5A) display a very similar behavior as the one observed for PaNTD (Figure 4).Nintedanib PLOS ONE | www.plosone.orgMutL N-Terminal Domain InterfacesFigure 2. Cluster analysis of MutL PaNTD and EcNTD. A) The central structure of the main cluster of ATP-bound PaNTD and EcNTD were compared. The corresponding ATP binding motives (I V) are colored (PaNTD in red; EcNTD in blue). B) Relative position in the superimposed structures mentioned of the conserved residue lysine of motif V (K307 in EcNTD, blue and 310 in PaNTD, red). doi:10.1371/journal.pone.0069907.gThis indicates that dimerization interface is less mobile for ADP and ATP bound BsNTD than ADP and ATP bound EcNTD. As for PaNTD, the region that possesses a similar Apo and ATP bound DRMSF with EcNTD corresponds to the ATP lid stem (residues ,750), but for the ATP lid a-helix a reduction in DRMSF can be observed for both PaNTD and BsNTD (residues 800). For TmNTD a model was constructed using EcNTD template (PDB:1B63A). It is observed that the apo form showed a more flexible structure than the ATP-bound form (Figure 5B). No differences were observed in rmsf values between ADP and ATPbound TmNTD (Figure 5B). The crystal structure of yeast PMS1 NTD bound to AMPPNP (PDB: 3H4LB) was used as an initial model. PMS1 apo form presented high RMSF values, particularly in the ATP lid region, where these values were 3 times larger than apo EcNTD (data not shown).PMID:24360118 More interesting, practically nodifferences in RMSF vales were shown when apo, ATP and ADPbound form were compared (Figure 5C).In vitro Assays showed that PaNTD is Dimeric when Bound to ADPSince MD simulations results indicated that there is a differential behavior of the dimerization interface of EcNTD and PaNTD, we analyzed the effect of such behavior on their oligomeric state. Ban Yang (1998) have shown by size-exclusion chromatography that binding of ADPnP induces a transition of EcNTD from monomer to dimer [12]. EcNTD dimerization is specific of ATP binding since no detectable changes are shown with ADP [15]. We cloned and purified recombinant PaNTD (residues 139 of PaMutL, monomeric MW = 37.7 kDa). When analyzed in SDSPAGE, PaNTD presented a MW ,40 kDa (data not shown). The oligomerization state of the apo protein was determined by gel filtration chromatography (Figure 6A). The elution profile of PaNTD presented two peaks, one corresponding to a MW of ,40 kDa similar to that e.