Xpected for the monomeric protein (MW:38 kDa) and a smaller one with an estimated molecular weight of ,70 kDa (Figure 6A, full black line). The peak relationship dimer:monomer was aprox. 0.2:0.8. The dimeric form of PaNTD is resistant to ON EDTA incubations as determined by native gels (Figure S4). PaNTD binding to ADP displaced monomer-dimer equilibrium as judged by size-exclusion chromatography (Figure 6A, dotted line). When incubated with ADP, the peak ratio inverted, and the dimeric form became the major species (Figure 6A). This result shows that, unlike EcNTD, PaNTD is capable of forming dimers in presence of ADP. No major differences were observed among the elution pattern of ADP or ATP bound PaNTD (Figure 6A). As a control, we cloned and purified his-tag EcNTD (residues 1- 342 of EcMutL, monomeric MW = 38.3 kDa) and the oligomerization state of the apo protein was determined by gel filtration chromatography (Figure 6B). The elution profiles of EcNTD in the apo form or bounded to ATP or ATP presented only one peak, corresponding to the monomeric protein (,40 kDa). Since EcNTD dimerization in presence of ATP is not evidenced in size exclusion chromatogFigure 3. Time evolution of ATP lid secondary structure in all atom MD.Evinacumab Secondary structure of the ATP lid from the different systems was analyzed. The secondary structure information was obtained using the do_dssp program (also see Figure S2) and representative structures were taken from the MD. Purple: alpha helix; blue: 3-helix; cyan: turn; white: coil. doi:10.1371/journal.pone.0069907.gPLOS ONE | www.plosone.orgMutL N-Terminal Domain InterfacesFigure 4. Analysis of PaNTD and EcNTD monomers residues mobility using structure based models (SBM). Root Mean Squeare Fluctuation (RMSF) difference between A) apo and ATP-bound monomer and B) ADP and ATP-bound monomer, for EcNTD (dotted line) and PaNTD (full black line) were calculated from SBM MD simulations.Alkaline phosphatase Dimerization interface (L1, L2, L3, L45 and ATP lid) is indicated with black horizontal bars. doi:10.1371/journal.pone.0069907.graphy, we performed cross-linking assays (Figure S5). EcNTD was incubated ON with EDTA, ADP, ATP and AMPPNP, and EcNTD dimers were only observed in presence of AMPPNP (Figure S5), as established [15]. These results show that, unlike EcNTD, PaNTD is capable of forming dimers in presence of ADP.Mix-solvent MD Simulations and in vitro Assays Allowed the Detection of PaNTD Protein-protein and Protein-DNA Interaction SitesNext, we aimed to determine the possible protein-protein and protein-DNA interaction sites that could mediate PaMutL activity regulation.PMID:27108903 The central structure of the main cluster for the apo and holo PaNTD obtained in the pure water simulations were used to perform a MD simulation in a solution of water with 20 isopropyl alcohol (iPrOH) [38]. The idea behind these simulations is that regions with an increased concentration in iPrOH predictPLOS ONE | www.plosone.orgregions of the protein that are more easily desolvated, indicating putative hot spots for interactions with other molecules. This method has recently been used to determine the interaction interface of a phospholipase with lipid membranes [43]. Spatial distribution functions (SDF) were calculated for PaNTD in both states (Figure 7A ). Also, the quantification of contacts along the MD between ATP-bound PaNTD Ca and iPrOH was included (Figure 7C). These analyses allowed the determination of PaNTD sites that preferentially bind iPrOH. T.