D above. Simulations were carried out for 50 ns. Spatial distribution function (SDF) was calculated using a bin of 2.5 A. VMD [39] was used for rendering, using an isosurface representation and a density isovalue of 20.MutL N-Terminal Domain InterfacesFigure 1. Sequence alignment of MutL PaNTD and EcNTD. ATP binding motifs conserved among GHKL ATPase superfamily (I V) are indicated. Red residues correspond to conserved residues within motifs. Loops involved in NTD dimerization (L1, L2, L3 and L45), as well as ATP lid, are indicated with horizontal bars. Amino acids that are identical (*), strongly similar (:) or weakly similar (.) are indicated. doi:10.1371/journal.pone.0069907.gAll simulations and analysis were performed using the GROMACS 4.0.7 simulation package (http://www.gromacs.org) [37]. VMD 1.8.7 [36] (http://www.ks.uiuc.edu/Research/vmd/) was used for visualization and figure rendering, and XMGRACE (http://plasma-gate.weizmann.ac.il/Grace/) was used for figure plotting.Results Molecular Dynamics Simulations show a Differential Effect of ATP Binding on E. coli and P. aeruginosa MutL ATP Lid DynamicsWe aim to determine if a differential behavior exists between Nterminal domains (NTD) of a MutL homologue that possesses with one that lacks of endonuclease activity. We hypothesized that NTD could behave differentially since the former have to cope with an additional activity. As mentioned before, ATP binding has been involved in the allosteric control of CTD activity [5,7,17,40]. Taking this into account, we performed all-atom molecular dynamics (MD) simulations of monomeric ATP-bound or ATPfree PaNTD and E. coli LN40 (here on denominated EcNTD). PaNTD amino acid sequence has an identity of 64 with EcNTD, and a similarity of 79 (Figure 1). Due to the lack of a crystal structure for PaNTD, a homology model was made using MODELLER [22] with EcNTD crystal structures bound to different nucleotides as templates (see Matherial and Methods). The PaNTD model can be superimposed with the crystal structure of EcNTD bound to AMPPNP (PDB: 1B63) with a RMSD value of 0.Pyridostigmine bromide 2 nm. Residues in the ATP binding site of EcNTD that directly bind the nucleotide are well conserved in PaNTD(Figure 1). Also, the four sequence motifs (I V) involved in nucleotide binding that are characteristic of GHKL ATPase family can be identified in the PaNTD sequence (Figure 1). According to sequence and structure alignment, putative PaNTD dimerization interface consists of L1 (residues 64, EcNTD r.J14 220.PMID:23892746 79 identity), L2 (r. 15466, EcNTD r. 15062. 85 identity), L3 (r. 30216, EcNTD 29913. 86 identity), L45 (r. 13035, EcNTD 12631. 86 identity) and ATP lid (r. 78101, EcNTD 747. 75 identity). MD simulations in explicit water were carried out for (i) the nucleotide free (apo) form of PaNTD, (ii) PaNTD complexed with ATP (holo) (iii) EcNTD in the apo form and (iv) holo EcNTD. All simulations were run for 200 ns. Given that the model for PaNTD is based on the holo form of EcNTD which is dimeric, and that the simulations of the apo state were generated by simple removal of the ligand, the simulations of the four systems started with a very similar global conformation. During the simulations we expected to observe deviations from the initial structures for two reasons. First, it has been suggested [15] that the final conformation of the protein is only reached when the protein dimerizes and the monomeric form may deviate from the initial structure. Second, when EcNTD is crysta.
