Analysis of the interaction interfaces of the N-terminal domain from Pseudomonas aeruginosa MutL

Mismatch Repair System corrects mutations arising from DNA replication that escape from DNA polymerase proofreading activity. This system consists of three main proteins, MutS-L-H, responsible for lesion recognition and repair. MutL is a member of GHKL ATPase family and its ATPase cycle has been proposed to modulate MutL activity during the repair process. Pseudomonas aeruginosa MutL (PaMutL) contains an N-terminal (NTD) ATPase domain connected by a linker to a C-terminal (CTD) dimerization domain that possesses metal ion-dependent endonuclease activity. With the aim to identify characteristics that allow the PaMutL NTD allosteric control of CTD endonuclease activity, we used an in silico and experimental approach to determine the interaction surfaces of P. aeruginosa NTD (PaNTD), and compared it with the well characterized Escherichia coli MutL NTD (EcNTD). Molecular dynamics simulations of PaNTD and EcNTD bound to or free of adenosine nucleotides showed that a significant difference exists between the behavior of the EcNTD and PaNTD dimerization interface, particularly in the ATP lid. Structure based simulations of MutL homologues with endonuclease activity were performed that allowed an insight of the dimerization interface behavior in this family of proteins. Our experimental results show that, unlike EcNTD, PaNTD is dimeric in presence of ADP. Simulations in mixed solvent allowed us to identify the PaNTD putative DNA binding patch and a putative interaction patch located opposite to the dimerization face. Structure based simulations of PaNTD dimer in presence of ADP or ATP suggest that nucleotide binding could differentially modulate PaNTD protein-protein interactions. Far western assays performed in presence of ADP or ATP are in agreement with our in silico analysis.